Echinocandine derivatives with antimicrobial activity

ABSTRACT

The invention relates to new polypeptide compounds represented by the following formula [I]:                    
     wherein 
     R 1  is hydrogen, etc, 
     R 2  is hydrogen, etc, 
     R 3  is hydrogen, etc, and 
     R 4  is hydrogen, etc, 
     or a salt thereof which have antimicrobial activities (especially, antifungal activities), inhibitory activity on β-1,3-glucan synthase, to process for preparation thereof, to a pharmaceutical composition comprising the same, and to a method for the prophylactic and/or therapeutic treatment of infectious diseases including  Pneumocystis carinii  infection (e.g.  Pneumocystis carinii  pneumonia) in a human being or an animal.

TECHNICAL FIELD

The present invention relates to new polypeptide compounds and saltsthereof which are useful as a medicament.

BACKGROUND ART

In U.S. Pat. Nos. 5,376,634, 5,502,033, etc., there are disclosed thepolypeptide compound and a salt thereof, which have antimicrobialactivities (especially antifungal activity).

DISCLOSURE OF INVENTION

The present invention relates to new polypeptide compounds (hereinafterreferred to as WF 738 derivative) and salts thereof.

More particularly, it relates to new polypeptide compound and saltsthereof, which have antimicrobial activities [especially, antifungalactivities, in which the fungi may include Asperaillus, Cryptococcus,Candida, Mucor, Actinomyces, Histoplasma, Dermatophyte, Malassezia,Fusarium and the like.], inhibitory activity on β-1,3-glucan synthase,and further which are expected to be useful for the prophylactic and/ortherapeutic treatment of Pneumocystis carinii infection (e.g.Pneumocystis carinii pneumonia) in a human being or an animal, to aprocess for preparation thereof, to a pharmaceutical compositioncomprising the same, and to a method for the prophylactic and/ortherapeutic treatment of infectious diseases including Pneumocystiscarinii infection (e.g. Pneumocystis carinii pneumonia) in a human beingor an animal.

The object polypeptide compound of the present invention is new and canbe represented by the following general formula [I]:

wherein

R¹ is hydrogen or acyl group,

R² is hydrogen or hydroxy,

R³ is hydrogen or methyl, and

R⁴ is hydrogen or hydroxy,

with proviso that

when R⁴ is hydroxy, then R² is hydroxy,

or a salt thereof.

The polypeptide compound [I] of the present invention can be prepared bythe processes as illustrated in the following schemes.

wherein

R_(a) ¹ is higher alkanoyl, and

R_(c) ¹ is acyl group exclusive of palmitoyl,

R², R³ and R⁴ are each as defined above.

Suitable salt of the object compound [I] is pharmaceutically acceptableand conventional non-toxic mono or di salt and include a metal salt suchas an alkali metal salt [e.g. sodium salt, potassium salt, etc.] and analkaline earth metal salt [e.g. calcium salt, magnesium salt, etc.], anammonium salt, an organic base salt [e.g. trimethylamine salt,triethylamine salt, N,N-diisopropylethylamine salt, pyridine salt,picoline salt, dicyclohexylamine salt, N,N-dibenzylethylenediamine salt,etc.], an organic acid addition salt [e.g. formate, acetate,trifluoroacetate, maleate, tartrate, methanesulfonate, benzenesulfonate,toluenesulfonate, etc.], an inorganic acid addition salt [e.g.hydrochloride, hydrobromide, hydroiodide, sulfate, phosphate, etc.], asalt with an amino acid [e.g. arginine salt, aspartic acid salt,glutamic acid salt, etc.], and the like.

In the above and subsequent descriptions of the present specification,suitable examples and illustration of the various definitions which thepresent invention intends to include within the scope thereof areexplained in detail as follows.

The term “lower” is intended to mean 1 to 6 carbon atom(s), unlessotherwise indicated.

The term “higher” is intended to mean 7 to 20 carbon atoms, unlessotherwise indicated.

The term “one or more” may be the number of 1 to 6, unless otherwiseindicated.

Suitable example of “halogen” may be fluoro, chloro, bromo, iodo, andthe like.

Suitable example of “lower alkoxy” may include straight or branched onesuch as methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy,tert-butoxy, pentyloxy, tert-pentyloxy, neo-pentyloxy, hexyloxy,isohexyloxy, and the like, in which the preferred one may be propoxy,pentyloxy and hexyloxy.

Suitable example of “higher alkoxy” may include straight or branched onesuch as heptyloxy, octyloxy, 3,5-dimethyloctyloxy, 3,7-dimethyloctyloxy,nonyloxy, decyloxy, undecyloxy, dodecyloxy, tridecyloxy, tetradecyloxy,hexadecyloxy, heptadecyloxy, octadecyloxy, nonadecyloxy, icosyloxy, andthe like, in which the preferred one may be (C₇-C₁₄)alkoxy, and the mostpreferred one may be octyloxy.

Suitable example of “lower alkyl” may include straight or branched onehaving 1 to 6 carbon atom(s), such as methyl, ethyl, propyl, isopropyl,butyl, isobutyl, sec-butyl, tert-butyl, pentyl, tert-pentyl, neo-pentyl,hexyl, isohexyl, and the like.

Suitable example of “higher alkyl” may include straight or branched onehaving 7 to 20 carbon atoms, such as heptyl, octyl, 3,5-dimethyloctyl,3,7-dimethyloctyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl,pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, icosyl, and thelike.

Suitable example of “aryl” and “ar” moiety may include phenyl which mayhave lower alkyl (e.g., phenyl, mesityl, tolyl, etc.), naphthyl,anthryl, and the like, in which the preferred one may be phenyl.

Suitable example of “aroyl” may include benzoyl, toluoyl, naphthoyl,anthrylcarbonyl, and the like, in which the preferred one may bebenzoyl.

Suitable example of “heterocyclic group” and “heterocyclic” moiety mayinclude

unsaturated 3 to 8-membered (more preferably 5 or 6-membered)heteromonocyclic group containing 1 to 4 nitrogen atom(s), for example,pyrrolyl, pyrrolinyl, imidazolyl, pyrazolyl, pyridyl, dihydropyridyl,pyrimidyl, pyrazinyl, pyridazinyl, triazolyl (e.g., 4H-1,2,4-triazolyl,1H-1,2,3-triazolyl, 2H-1,2,3-triazolyl, etc.), tetrazolyl (e.g.,1H-tetrazolyl, 2H-tetrazolyl, etc.), etc.;

saturated 3 to 8-membered (more preferably 5 or 6-membered)heteromonocyclic group containing 1 to 4 nitrogen atom(s), for example,pyrrolidinyl, imidazolidinyl, piperidyl, piperazinyl, etc.;

unsaturated condensed heterocyclic group containing 1 to 4 nitrogenatom(s), for example, indolyl, isoindolyl, indolinyl, indolizinyl,benzimidazolyl, quinolyl, isoquinolyl, indazolyl, benzotriazolyl, etc.;

unsaturated 3 to 8-membered (more preferably 5 or 6-membered)heteromonocyclic group containing 1 to 2 oxygen atom(s) and 1 to 3nitrogen atom(s), for example, oxazolyl, isoxazolyl, oxadiazolyl (e.g.,1,2,4-oxadiazolyl, 1,3,4-oxadiazolyl, 1,2,5-oxadiazolyl, etc.), etc.;

saturated 3 to 8-membered (more preferably 5 or 6-membered)heteromonocyclic group containing 1 to 2 oxygen atom(s) and 1 to 3nitrogen atom(s), for example, morpholinyl, sydnonyl, etc.;

unsaturated condensed heterocyclic group containing 1 to 2 oxygenatom(s) and 1 to 3 nitrogen atom(s), for example, benzoxazolyl,benzoxadiazolyl, etc.;

unsaturated 3 to 8-membered (more preferably 5 or 6-membered)heteromonocyclic group containing 1 to 2 sulfur atom(s) and 1 to 3nitrogen atom(s), for example, thiazolyl, isothiazolyl, thiadiazolyl(e.g., 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,3,4-thiadiazolyl,1,2,5-thiadiazolyl, etc.), dihydrothiazinyl, etc.;

saturated 3 to 8-membered (more preferably 5 or 6-membered)heteromonocyclic group containing 1 to 2 sulfur atom(s) and 1 to 3nitrogen atom(s) for example, thiazolidinyl, etc.;

unsaturated 3 to 8-membered (more preferably 5 or 6-membered)heteromonocyclic group containing 1 to 2 sulfur atom(s), for example,thienyl, dihydrodithiinyl, dihydrodithionyl, etc.;

unsaturated condensed heterocyclic group containing 1 to 2 sulfuratom(s) and 1 to 3 nitrogen atom(s), for example, benzothiazolyl,benzothiadiazolyl, imidazothiadiazolyl, etc.;

unsaturated 3 to 8-membered (more preferably 5 or 6-membered)heteromonocyclic group containing an oxygen atom, for example, furyl,etc.;

saturated 3 to 8-membered (more preferably 5 or 6-membered)heteromonocyclic group containing an oxygen atom, for example,tetrahydrofuran, tetrahydropyran, etc.;

unsaturated 3 to 8-membered (more preferably 5 or 6-membered)heteromonocyclic group containing an oxygen atom and 1 to 2 sulfuratom(s), for example, dihydrooxathiinyl, etc.;

unsaturated condensed heterocyclic group containing 1 to 2 sulfuratom(s), for example, benzothienyl, benzodithiinyl, etc.;

unsaturated condensed heterocyclic group containing an oxygen atom and 1to 2 sulfur atom(s), for example, benzoxathiinyl, etc.; and the like.

Suitable example of “acyl group” may include aliphatic acyl, aromaticacyl, heterocyclic acyl, arylaliphatic acyl and heterocyclic-aliphaticacyl derived from carboxylic acid, carbonic acid, carbamic acid,sulfonic acid, and the like.

Suitable example of the “acyl group” thus explained may be:

lower alkanoyl [e.g. formyl, acetyl, propionyl, butyryl, isobutyryl,valeryl, hexanoyl, pivaloyl, etc.] which may have one or more(preferably 1 to 3) suitable substituent(s) such as halogen, aryl whichmay have one or more (preferably 1 to 3) suitable substituent(s) such ashydroxy; higher alkoxy; aryl; or the like, lower alkoxy, amino,protected amino, (preferably, acylamino) such as loweralkoxycarbonylamino (e.g. methoxycarbonylamino, ethoxycarbonylamino,propoxycarbonylamino, butoxycarbonylamino, t-butoxycarbonylamino,pentyloxycarbonylamino, hexyloxycarbonylamino, etc.);ar(lower)alkoxycarbonylamino such as phenyl(lower)alkoxycarbonylamino(e.g. benzyloxycarbonylamino, etc.); or the like, di(lower)alkylamino(e.g. dimethylamino, N-methylethylamino, diethylamino,N-propylbutylamino, dipentylamino, dihexylamino, etc.), loweralkoxyimino (e.g. methoxyimino, ethoxyimino, propoxyimino, butoxyimino,t-butoxyimino, pentyloxyimino, hexyloxyimino, etc.),ar(lower)alkoxyimino such as phenyl(lower)alkoxyimino (e.g.benzyloxyimino, phenethyloxyimino, benzhydryloxyimino, etc.) which mayhave one or more (preferably 1 to 3) suitable substituent(s) like higheralkoxy, or the like, heterocyclicthio (preferably, pyridylthio) whichmay have one or more (preferably 1 to 3) suitable substituent(s) likehigher alkyl, heterocyclic group which may have one or more (preferably1 to 3) suitable substituent(s) such as amino; aforesaid protectedamino; higher alkyl; or the like, or the like,

higher alkanoyl [e.g. heptanoyl, octanoyl, nonanoyl, decanoyl,undecanoyl, lauroyl, tridecanoyl, myristoryl, pentadecanoyl, palmitoyl,10,12-dimethyltetradecanoyl, heptadecanoyl, stearoyl, nonadecanoyl,icosanoyl, etc.], in which the preferred one may be (C₇-C₁₇)alkanoyl,and the most preferred one may be palmitoyl,

lower alkenoyl [e.g. acryloyl, methacryloyl, crotonoyl, 3-pentenoyl,5-hexenoyl, etc.] which may have one or more (preferably 1 to 3)suitable substituent(s) such as aryl which may have one or more(preferably 1 to 3) suitable substituent(s) like higher alkoxy, or thelike, higher alkenoyl [e.g. 4-heptenoyl, 3-octenoyl, 3,6-decadienoyl,3,7,11-trimethyl-2,6,10-dodecatrienoyl, 4,10-heptadecadienoyl, etc.],

lower alkoxycarbonyl [e.g. methoxycarbonyl, ethoxycarbonyl,propoxycarbonyl, butoxycarbonyl, t-butoxycarbonyl, pentyloxycarbonyl,hexyloxycarbonyl, etc.],

higher alkoxycarbonyl [e.g. heptyloxycarbonyl, octyloxycarbonyl,2-ethylhexyloxycarbonyl, nonyloxycarbonyl, decyloxycarbonyl,3,7-dimethyloctyloxycarbonyl, undecyloxycarbonyl, dodecyloxycarbonyl,tridecyloxycarbonyl, tetradecyloxycarbonyl, pentadecyloxycarbonyl,3-methyl-10-ethyldodecyloxycarbonyl, hexadecyloxycarbonyl,heptadecyloxycarbonyl, octadecyloxycarbonyl, nonadecyloxycarbonyl,icosyloxycarbonyl, etc.],

aryloxycarbonyl [e.g. phenoxycarbonyl, naphthyloxycarbonyl, etc.],

arylglyoxyloyl [e.g. phenylglyoxyloyl, naphthylglyoxyloyl, etc.],

ar(lower)alkoxycarbonyl which may have one or more suitable substituents) like phenyl(lower)alkoxycarbonyl which may have nitro or lower alkoxy[e.g. benzyloxycarbonyl, phenethyloxycarbonyl, p-nitrobenzyloxycarbonyl,p-methylbenzyloxycarbonyl, etc.],

lower alkylsulfonyl [e.g. methylsulfonyl, ethylsulfonyl, propylsulfonyl,isopropylsulfonyl, pentylsulfonyl, butylsulfonyl, etc.],

arylsulfonyl [e.g. phenylsulfonyl, naphthylsulfonyl, etc.] which mayhave one or more (preferably 1 to 3) suitable substituent(s) such aslower alkyl, higher alkoxy, or the like,

ar(lower)alkylsulfonyl such as phenyl(lower)alkylsulfonyl [e.g.benzylsulfonyl, phenethylsulfonyl, benzhydrylsulfonyl, etc.], or thelike,

aroyl which may have one or more (preferably 1 to 5) suitablesubstituent(s) such as halogen, lower alkyl, higher alkyl, lower alkoxywhich may have one or more (preferably 1 to 10) suitable substituent(s)such as lower alkoxy; halogen; aryl; or the like, higher alkoxy whichmay have one or more (preferably 1 to 17) suitable substituent(s) likehalogen, or the like, and the like, in which the preferred one may bearoyl having higher alkoxy,

aroyl substituted with heterocyclic group which has aryl having loweralkoxy,

aroyl substituted with heterocyclic group which has aryl having higheralkoxy,

ar(lower)alkenoyl substituted with aryl having lower alkoxy,

ar(lower)alkenoyl substituted with aryl having higher alkoxy,

aroyl substituted with aryl which has aryl having lower alkoxy,

aroyl substituted with aryl which has aryl having higher alkoxy,

aroyl substituted with heterocyclic group which has aryl. substitutedwith aryl having lower alkoxy,

aroyl substituted with heterocyclic group which has aryl substitutedwith aryl having higher alkoxy.

Suitable example of “aroyl having higher alkoxy” may be benzoyl having(C₇-C₁₇)alkoxy, in which the preferred one may be benzoyl havingoctyloxy.

Suitable example of “aroyl substituted with heterocyclic group which hasaryl having lower alkoxy” may be benzoyl substituted with saturated6-membered heteromonocyclic group containing 1 to 4 nitrogen atom(s)which has phenyl having (C₄-C₆)alkoxy, benzoyl substituted withunsaturated 5-membered heteromonocyclic group containing 1 to 2 sulfuratom(s) and 1 to 3 nitrogen atom(s) which has phenyl having(C₄-C₆)alkoxy, benzoyl substituted with unsaturated condensedheterocyclic group containing 1 to 2 sulfur atom(s) and 1 to 3 nitrogenatom(s) which has phenyl having (C₄-C₆)alkoxy or benzoyl substitutedwith unsaturated 5-membered heteromonocyclic group containing 1 to 2oxygen atom(s) and 1 to 3 nitrogen atom(s) which has phenyl having(C₄-C₆)alkoxy, in which the preferred one may be benzoyl substitutedwith piperazinyl which has phenyl having (C₄-C₆)alkoxy, benzoylsubstituted with thiadiazolyl which has phenyl having (C₄-C₆)alkoxy,benzoyl substituted with thiazolyl which has phenyl having(C₄-C₆)alkoxy, benzoyl substituted with imidazothiadiazolyl which hasphenyl having (C₄-C₆)alkoxy or benzoyl substituted with isoxazolyl whichhas phenyl having (C₄-C₆)alkoxy, and the most preferred one may bebenzoyl substituted with piperazinyl which has phenyl having hexyloxy,benzoyl substituted with thiadiazolyl which has phenyl having hexyloxy,benzoyl substituted with thiazolyl which has phenyl having pentyloxy orhexyloxy, benzoyl substituted with imidazothiadiazolyl which has phenylhaving pentyloxy or benzoyl substituted with isoxazolyl which has phenylhaving pentyloxy.

Suitable example of “ar(lower)alkenoyl substituted with aryl havinglower alkoxy” may be phenyl(C₃-C₆)alkenoyl substituted with phenylhaving (C₄-C₆)alkoxy, in which the preferred one may be phenylacryloylsubstituted with phenyl having pentyloxy.

Suitable example of “aroyl substituted with aryl which has aryl havinglower alkoxy” may be benzoyl substituted with phenyl which has phenylhaving (C₄-C₆)alkoxy, in which the preferred one may be benzoylsubstituted with phenyl which has phenyl having pentyloxy.

Suitable example of “aroyl substituted with heterocyclic group which hasaryl substituted with aryl having lower alkoxy” may be benzoylsubstituted with unsaturated 5-membered heteromonocyclic groupcontaining 1 to 2 sulfur atom(s) and 1 to 3 nitrogen atom(s) which hasphenyl substituted with phenyl having (C₁-C₄)alkoxy, in which thepreferred one may be benzoyl substituted with thiadiazoyl which hasphenyl substituted with phenyl having (C₁-C₄)alkoxy or benzoylsubstituted with thiazolyl which has phenyl substituted with phenylhaving (C₁-C₄)alkoxy, and the most preferred one may be benzoylsubstituted with thiadiazolyl which has phenyl substituted with phenylhaving propoxy or benzoyl substituted with thiazoyl which has phenylsubstituted with phenyl having propoxy.

The process for preparing the object compound [I] or a salt thereof ofthe present invention is explained in detail in the following.

Process 1

The object compound [Ia] or a salt thereof can be prepared by thefermentation process.

The fermentation process is explained in detail in the following.

The compound [Ia] or a salt thereof of this invention can be produced byfermentation of the compound [Ia] or a salt thereof-producing strainbelonging to the genus Coleophoma such as Coleophoma crateriformisNo.738 in a nutrient medium.

(i) Microorganism

Particulars of the microorganism used for producing the compound [Ia] ora salt thereof is explained in the following.

The fungus strain No.738 was originally isolated from a leaf sample,collected at Mt. Tateyama, Kaminiikawa-gun, Toyama-ken, Japan. Thisorganism grew rather restrictedly on various culture media, and formedgrayish colonies. The strain produced pycnidial conidiomata flattened atthe base, on steam-sterilized leaf segments affixed on an agar medium byinoculating the isolate, while it formed neither teleomorph nor anamorphon agar media. The conidiomata were discoid or sometimes papillate, darkbrown to black, and formed conidiophores on the lower cells of its innerwalls. Conidiogenous cells were ampulliform to lageniform, and conidiawere hyaline, one-celled, cylindrical. They were covered withthin-walled sheath. On the basis of comparing the morphologicalcharacteristics with fungal taxonomic criteria by von Arx (J.A. van Arx:The Genera of Fungi—Sporulating in Pure Culture. 3rd ed., pp.145-163, J.Cramer, Vaduz, 1974), strain No.738 was considered to belong to thecoelomycete genus Coleophoma Höhn. 1907 (Sphaeropsidales). Itsmycological characteristics were as follows.

Cultural characteristics on various agar media are summarized inTable 1. Culture on potato dextrose agar grew rather rapidly, attaining3.0-4.0 cm in diameter two weeks later at 25° C. This colony surface wasconvex to raised, cottony and partly fasciate, sectoring, and showedseveral colors; pale gray to olive at the center, white to yellowishwhite at the margin and olive brown at the sectors. The colony marginwas wet and lustrous. The reverse was yellowish gray, and olive gray atthe sectors. Conidial structures were not observed. Colonies on cornmeal agar grew rather restrictedly, attaining 2.5-3.5 cm in diameterunder the same conditions. The surface was flat, felty and dark gray toolive gray. The colony margin was submerged, lustrous and olive. Thereverse was black, and olive gray at the margin. Conidial structureswere not produced.

The morphological characteristics were determined from the cultures onsterile leaf segments affixed on a Miura' LCA plate (Miura, K. and M.Kudo: Trans. Mycol. Soc. Japan, 11:116-118, 1970). Conidiomata formed onthe leaf segments alone. They were pycnidial, superficial, separate,discoid or sometimes papillate none-ostiolate or indistinctly ostiolate,flattened at the base, unilocular, thick-walled textura angularis withthin upper wall, dark brown to black, 90-160 (−400) mm in diameter and50-90 mm high. Conidiophores formed from the lower cells of innerpycnidial walls. The conidiophore-producing cells were hyaline,subglobose and 4-6 (−7.5) mm in diameter. The conidiophores werehyaline, smooth, septate, simple to sparingly branched, and 3-13.5×3-4mm. They formed discrete conidiogenous cells at the apex. Theconidiogenous cells were hyaline, smooth, ampulliform to lageniform, orcylindrical, and 4-8.5×2.5-4 mm. Conidia were hyaline, smooth,one-celled, cylindrical, rounded at the apical end, with a smallprojection at the base, and 10-13×2-3 mm. Both conidium andconidiogenous cell were covered with a large sheath. The sheaths werehyaline, thin-walled campanulate to cylindrical and 14-21.5×3-5 mm.Vegetative hyphae were smooth, septate, brown and branched. The hyphalcells were cylindrical, and 2-7 mm in width. Chlamydospores were notobserved.

Strain No.738 was able to grow at the temperature range from 3 to 30° C.with the growth optimum at 20 to 24° C. These temperature data weredetermined on potato dextrose agar (made by NISSUI).

According to the taxonomic criteria of the genus Coleophoma by Sutton(B. C. Sutton: The Coelomycetes—Fungi Imperfecti with Pycnidia, Acervuliand Stroma, pp.401-403, Commonwealth Mycological Institute, Kew, 1980),the strain No.738 resembles Coleophoma crateriformis (Dur. & Mont.)Hohn. 1907. Moreover, the above characteristics corresponded with thedescription by Sutton, with a few exceptions: superficial,none-ostiolate or indistinctly ostiolate conidiomata and sheathscovering with conidiogenous cells and conidia. However, the lattercharacteristics, sheaths, were described as paraphyses by Sutton. Thus,we identified the strain as one strain of Coleophoma crateriformis, andnamed it Coleophoma crateriformis No.738. This strain has been depositedwith the National Institute of Bioscience and Human-Technology, Agencyof Industrial Science and Technology, (1-3, Higashi 1-chome,Tsukuba-shi, IBARAKI 305 JAPAN) as FERM BP-5796 (deposited date: Jan.23, 1997).

TABLE 1 Cultural characteristics of strain No.738. Media Culturalcharacteristics Malt extract agar* G: Rather restrictedly, 3.0-3.5 cm S:Circular, flat, felty, exudate at the center, formed no anamorph, darkgray (1F1) to violet gray (16F2) at the center and yellowish white (4A2)to yellowish gray (4B2) at the margin R: Dark gray (1F1) at the centerand yellowish gray (4B2) to olive gray (4D3) at the margin Potatodextrose G: Rather rapidly, 3.0-4.0 cm agar (Difco 0013) S: Circular toirregular, convex to raised, cottony and partly fasciate, wet andlustrous at the margin, sectoring, formed no anamorph, pale gray (1B1)to olive (1F3) at the center, white to yellowish white (4A2) at themargin and olive brown (4F3) at the sectors R: Yellowish gray (2B-C2),and olive gray (2F2) at the sectors Czapek's solution G: Veryrestrictedly, 0.5 cm agar* S: Irregular, scanty, flat, formed noanamorph, grayish brown (5F3) R: Brownish gray (5F2) Sabouraud dextroseG: Rather restrictedly, 3.0-3.5 cm agar (Difco 0190) S: Circular,convex, felty and partly fasciate, wet, lustrous, sectoring, formed noanamorph, yellowish white (4A2), and grayish brown (5D-E3) at the centerand sectors R: Pale yellow (4A3), and olive brown (4D3) at the sectorsEmerson Yp Ss agar G: Restrictedly, 1.5-2.5 cm (Difco 0739) S: Circularto irregular, flat to raised; felty, sulcate, sectoring, formed noanamorph, pale gray (1B1) to light gray (1D1), produced dark greensoluble pigment R: Olive (1-2F3), and yellowish gray (2D2) at the centerCorn meal agar G: Rather restrictedly, 2.5-3.5 cm (Difco 0386) S:Circular, flat, felty, submerged at the margin, lustrous, formed noanamorph, dark gray (1F1) to olive gray (2F2), and olive (1E- F4) at themargin R: Black, and olive gray (2E2) a the margin MY20 agar* G: Ratherrestrictedly, 2.5-3.5 cm S: Circular to irregular, flat, felty, wet,sectoring, formed no anamorph, olive brown (4D4-4F3), and grayish orange(6B3) at the center R: Olive brown (4E4), and pale orange (5A3) at thecenter Abbreviation G: growth, measuring colony size in diameter, S:colony surface and R: reverse. *The compositions of malt extract agar,Czapek's solution agar and MY20 agar were based on JCM Catalogue ofStrains (Nakase, T., 5th ed., 503p., Japan Collection of Microorganismsand Life Science Research Information Section of the Institute ofPhysical and Chemical Research, Saitama, 1992).

These characteristics were observed after 14 days of incubation at 25°C. The color descriptions were based on Methuen Handbook of Colour(Kornerup, A. and J. H. Wanscher, 3rd ed., 525p., Methuen, London,1978).

(ii) Production of the compound [Ia] or a salt thereof

The compound [Ia] or a salt thereof of this invention is produced whenthe compound [Ia] or a salt thereof-producing strain belonging to thegenus Coleophoma is grown in a nutrient medium containing sources ofassimilable carbon and nitrogen under aerobic conditions (e.g. shakingculture, submerged culture, etc.).

The preferred sources of carbon in the nutrient medium are carbohydratessuch as glucose, sucrose, starch, fructose or glycerin, or the like.

The preferred sources of nitrogen are yeast extract, peptone, glutenmeal, cotton seed flour, soybean meal, corn steep liquor, dried yeast,wheat germ, etc., as well as inorganic and organic nitrogen compoundssuch as ammonium salts (e.g. ammonium nitrate, ammonium sulfate,ammonium phosphate, etc.), urea or amino acid, or the like.

The carbon and nitrogen sources, though advantageously employed incombination, need not to be used in their pure form because less purematerials, which contain traces of growth factors and considerableguantities of mineral nutrients, are also suitable for use.

When desired, there may be added to the medium mineral salts such assodium or calcium carbonate, sodium or potassium phosphate, sodium orpotassium chloride, sodium or potassium iodide, magnesium salts, coppersalts, zinc salts, or cobalt salts, or the like.

If necessary, especially when the culture medium foams seriously adefoaming agent, such as liquid paraffin, fatty oil, plant oil, mineraloil or silicone, or the like may be added.

As in the case of the preferred methods used for the production of otherbiologically active substances in massive amounts, submerged aerobiccultural conditions are preferred for the production of the compound[Ia] or a salt thereof in massive amounts.

For the production in small amounts, a shaking or surface culture in aflask or bottle is employed.

Further, when the growth is carried out in large tanks, it is preferableto use the vegetative form of the organism for inoculation in theproduction tanks in order to avoid growth lag in the process ofproduction of the compound [Ia] or a salt thereof. Accordingly, it isdesirable first to produce a vegetative inoculum of the organism byinoculating a relatively small quantity of culture medium with spores ormycelia of the organism and culturing said inoculated medium, and thento transfer the cultured vegetative inoculum to large tanks. The medium,in which the vegetative inoculum is produced, is substantially the sameas or different from the medium utilized for the production of thecompound [Ia] or a salt thereof.

Agitation and aeration of the culture mixture may be accomplished in avariety of ways. Agitation may be provided by a propeller or similarmechanical agitation equipment, by revolving or shaking the fermentor,by various pumping equipment or by the passage of sterile air throughthe medium. Aeration may be effected by passing sterile air through thefermentation mixture.

The fermentation is usually conducted at a temperature between about 10°C. and 40° C., preferably 20° C. to 30° C., for a period of about 50hours to 150 hours, which may be varied according to fermentationconditions and scales.

When the fermentation is completed, the culture broth is then subjectedfor recovery of the compound [Ia] or a salt thereof to variousprocedures conventionally used for recovery and purification ofbiological active substances, for instance, solvent extraction with anappropriate solvent or a mixture of some solvents, chromatography orrecrystallization from an appropriate solvent or a mixture of somesolvents, or the like.

According to this invention, in general, the compound [Ia] or a saltthereof is found both in the cultured mycelia and cultured broth.Accordingly, then the compound [Ia] or a salt thereof is removed fromthe whole broth by means of extraction using an appropriate organicsolvent such as acetone or ethyl acetate, or a mixture of these solvent,or the like.

The extract is treated by a conventional manner to provide the compound[Ia] or a salt thereof, for example, the extract is concentrated byevaporation or distillation to a smaller amount and the resultingresidue containing active material, i.e. the compound [Ia] or a saltthereof is purified by conventional purification procedures, forexample, chromatography on recrystallization from an appropriate solventor a mixture of some solvents.

When the object compound is isolated as a salt of the compound [Ia], itcan be converted to the free compound [Ia] or another salt of thecompound [Ia] according to a conventional manner.

Process 2

The object polypeptide compound [Ib] or a salt thereof can be preparedby subjecting a compound [Ia] or a salt thereof to elimination reactionof N-acyl group.

This reaction is carried out in accordance with a conventional methodsuch as hydrolysis, reduction, reaction with an enzyme or the like.

The hydrolysis is preferably carried out in the presence of a base or anacid including Lewis acid. Suitable base may include an inorganic baseand an organic base such as an alkali metal [e.g. sodium, potassium,etc.], an alkaline earth metal [e.g. magnesium, calcium, etc.], thehydroxide or carbonate or bicarbonate thereof, trialkylamine [e.g.trimethylamine, triethylamine, etc.], picoline,1,5-diazabicyclo[4.3.0]non-5-ene, 1,4-diazabicyclo[2.2.2]octane,1,8-diazabicyclo[5.4.0]undec-7-ene, or the like.

Suitable acid may include an organic acid [e.g. formic acid, aceticacid, propionic acid, trichloroacetic acid, trifluoroacetic acid, etc.]and an inorganic acid [e.g. hydrochloric acid, hydrobromic acid,sulfuric acid, hydrogen chloride, hydrogen bromide, etc.]. Theelimination using Lewis acid such as trihaloacetic acid [e.g.trichloroacetic acid, trifluoroacetic acid, etc.], or the like, ispreferably carried out in the presence of cation trapping agents [e.g.anisole, phenol, etc.].

The reaction is usually carried out in a solvent such as water, analcohol [e.g. methanol, ethanol, etc.], methylene chloride,tetrahydrofuran, a mixture thereof or any other solvent which does notadversely influence the reaction. A liquid base or acid can be also usedas the solvent. The reaction temperature is not critical and thereaction is usually carried out under cooling to warming.

The reduction method applicable for the elimination reaction may includechemical reduction and catalytic reduction.

Suitable reducting agents to be used in chemical reduction are acombination of metal [eg.. tin, zinc, iron, etc.] or metallic compound[e.g. chromium chloride, chromium acetate, etc.] and an organic orinorganic acid [e.g. formic acid, acetic acid, propionic acid,trifluoroacetic acid, p-toluenesulfonic acid, hydrochloric acid,hydrobromic acid, etc.].

Suitable catalysts to be used in catalytic reduction are conventionalones such as platinum catalysts [e.g. platinum plate, spongy platinum,platinum black, colloidal platinum, platinum oxide, platinum wire,etc.], palladium catalysts [e.g. spongy palladium, palladium black,palladium oxide, palladium on carbon, colloidal palladium, palladium onbarium sulfate, palladium on barium carbonate, etc.], nickel catalysts[e.g. reduced nickel, nickel oxide, Raney nickel, etc.], cobaltcatalysts [e.g. reduced cobalt, Raney cobalt, etc.], iron catalysts[e.g. reduced iron, Raney iron, etc.], copper catalysts [e.g. reducedcopper, Raney copper, Ullman copper, etc.] and the like.

The reduction is usually carried out in a conventional solvent whichdoes not adversely influence the reaction such as water, methanol,ethanol, propanol, N,N-dimethylformamide, or a mixture thereof.Additionally, in case that the above-mentioned acids to be used inchemical reduction are in liquid, they can also be used as a solvent.Further, a suitable solvent to be used in catalytic reduction may be theabove-mentioned solvent, and other conventional solvent such as diethylether, dioxane, tetrahydrofuran, etc., or a mixture thereof.

The reaction temperature of this reduction is not critical and thereaction is usually carried out under cooling to warming.

The reaction with an enzyme can be carried out by reacting the compound[Ia] or a salt thereof with an enzyme suitable for the eliminationreaction of N-acyl group.

Suitable example of said enzyme may include the one produced by certainmicroorganisms of the Streptomycetaceae, the Actinoplanaceae, theOidiodendron or the Verticillium, for example, Streptomyces sp. No.6907(FERM BP-5809), Streptomyces anulatus No.4811 (FERM BP-5808),Streptomyces anulatus No.8703 (FERM BP-5810), Actinoplanes utahensisIFO-13244, Actinoplanes utahensis ATCC 12301, Actinoplanes missenriensesNRRL 12053, Oidiodendron sp. No.30084 (FERM P-15550), Verticillium sp.No.30085 (FERM P-15551), or the like; and the like.

This elimination reaction is usually carried out in a solvent such asphosphate buffer, Tris-HCl buffer or any other solvent which does notadversely influence the reaction.

The reaction temperature is not critical and the reaction can be carriedout at room temperature or under warming.

Process 3

The object polypeptide compound [Ic] or a salt thereof can be preparedby reacting the compound [Ib] or its reactive derivative at the aminogroup or a salt thereof with the compound [III] or its reactivederivative at the carboxy group or a salt thereof.

Suitable reactive derivative at the carboxy group of the compound [III]may include an acid halide, an acid anhydride, an activated amide, anactivated ester, and the like. Suitable examples of the reactivederivatives may be an acid chloride; an acid azide; a mixed acidanhydride with an acid such as substituted phosphoric acid [e.g.,dialkylphosphoric acid, phenylphosphoric acid, diphenylphosphoric acid,dibenzylphosphoric acid, halogenated phosphoric acid, etc.],dialkylphosphorous acid, sulfurous acid, thiosulfuric acid, sulfuricacid, sulfonic acid [e.g., methanesulfonic acid, etc.], aliphaticcarboxylic acid [e.g., acetic acid, propionic acid, butyric acid,isobutyric acid, pivaric acid, pentanoic acid, isopentanoic acid,2-ethylbutyric acid, trichloroacetic acid, etc.]; or aromatic carboxylicacid [e.g., benzoic acid, etc.]; a symmetrical acid anhydride; anactivated amide with imidazole, 4-substituted imidazole,dimethylpyrazole, triazole, tetrazole or 1-hydroxy-1H-benzotriazole; oran activated ester [e.g., cyanomethyl ester, methoxymethyl ester,dimethyliminomethyl

ester, vinyl ester, propargyl ester, p-nitrophenyl ester,2,4-dinitrophenyl ester, trichlorophenyl ester, pentachlorophenyl ester,mesylphenyl ester, phenylazophenyl ester, phenyl thioester,p-nitrophenyl thioester, p-cresyl thioester, carboxymethyl thioester,pyranyl ester, pyridyl ester, piperidyl ester, 8-quinolyl thioester,etc.], or an ester with a N-hydroxy compound [e.g.N,N-dimethylhydroxylamine, 1-hydroxy-2-(1H)-pyridone,N-hydroxysuccinimide, N-hydroxyphthalimide, 1-hydroxy-1H-benzotriazole,etc.], and the like. These reactive derivatives can optionally beselected from them according to the mind of the compound [III] to beused.

Suitable salts of the compound [III] and its reactive derivative can bereferred to the ones as exemplified for the object polypeptide compound[I].

The reaction is usually carried out in a conventional solvent such aswater, alcohol [e.g., methanol, ethanol, etc.], acetone, dioxane,acetonitrile, chloroform, methylene chloride, ethylene chloride,tetrahydrofuran, ethyl acetate, N,N-dimethylformamide, pyridine or anyother organic solvent which does not adversely influence the reaction.These conventional solvent may also be used in a mixture with water.

In this reaction, when the compound [III] is used in a free acid form orits salt form, the reaction is preferably carried out in the presence ofa conventional condensing agent such as N,N′-dicyclohexylcarbodiimide;N-cyclohexyl-N′-morpholinoethylcarbodiimide;N-cyclohexyl-N′-(4-diethylaminocyclohexyl)carbodiimide;N,N′-diethylcarbodiimide, N,N′-diisopropylcarbodiimide;N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide,N,N-carbonylbis-(2-methylimidazole);pentamethyleneketene-N-cyclohexylimine;diphenylketene-N-cyclohexylimine; ethoxyacetylene;1-alkoxy-2-chloroethylene; trialkyl phosphite; ethyl polyphosphate;isopropyl polyphosphate; phosphorus oxychloride (phosphoryl chloride);phosphorus trichloride; thionyl chloride; oxalyl chloride; lower alkylhaloformate [e.g., ethyl chloroformate, isopropyl chloroformate, etc.];triphenylphosphine; 2-ethyl-7-hydroxybenzisoxazolium salt;2-ethyl-5-(m-sulfophenyl)isoxazolium hydroxide intramolecular salt;1-(p-chlorobenzenesulfonyloxy)-6-chloro-1H-benzotriazole; so-calledVilsmeier reagent prepared by the reaction of N,N-dimethylformamide withthionyl chloride, phosgene, trichloromethyl chloroformate, phosphorousoxychloride, methanesulfonyl chloride, etc.; or the like.

The reaction may also be carried out in the presence of an inorganic ororganic base such as an alkali metal carbonate, alkali metalbicarbonate, tri(lower)alkylamine, pyridine, di(lower)alkylaminopyridine(e.g., 4-dimethylaminopyridine, etc.), N-(lower)alkylmorpholine,N,N-di(lower)alkylbenzylamine, or the like.

The reaction temperature is not critical, and the reaction is usuallycarried out under cooling to warming.

The compounds obtained by the above Processes 1 to 3 can be isolated andpurified by a conventional method such as pulverization,recrystallization, column-chromatography, high-performance liquidchromatography (HPLC), reprecipitation, or the like.

The compounds obtained by the above Processes 1 to 3 may be obtained asits hydrate, and its hydrate is included within the scope of thisinvention.

BIOLOGICAL PROPERTY OF THE POLYPEPTIDE COMPOUND [I] OF THE PRESENTINVENTION

In order to show the usefulness of the polypeptide compound [I] of thepresent invention, the biological data of the representative compound isexplained in the following.

Test (Antimicrobial Activity)

Test Method

Antimicrobial activity of the object compounds of Examples 1 and 2 weredetermined by a serial broth dilution method using 96-well microtiterplate in 100 μl of MEM (Eagle's minimum essential medium) for Candidaalbicans and in 100 μl of yeast nitrogen base dextrose medium forAspergillus fumigatus. The inoculum was adjusted to 1×10⁵ colony formingunits/ml. Candida albicans and Asperaillus fumigatus were cultured at37° C. for 24 hours in 5% CO₂ incubator. After incubation, the growthinhibition of microorganisms in each well was determined by microscopicobservation. The results were shown as MEC (minimum effectiveconcentration : μg/ml) value (Table 2).

Test Result

TABLE 2 [MEC (μg/ml)] Test compound Microorganisms WF 738A WF 738C WF738B Candida albicans 0.04 0.04 0.04 FP633 Aspergillus 0.04 0.04 0.04fumigatus FP1305

From the test result, it is realized that the object polypeptidecompound [I] of the present invention has an antimicrobial activity(especially, antifungal activity).

The pharmaceutical composition of the present invention can be used inthe form of a pharmaceutical preparation, for example, in solid,semisolid or liquid form, which contains the object polypeptide compound[I] or a pharmaceutically acceptable salt thereof, as an activeingredient in admixture with an organic or inorganic carrier orexcipient which is suitable for rectal; pulmonary (nasal or buccalinhalation); ocular; external (topical); oral administration; parenteral(including subcutaneous, intravenous and intramuscular) administrations;insufflation (including aerosols from metered dose inhalator);nebulizer; or dry powder inhalator.

The active ingredient may be compounded, for example, with the usualnon-toxic, pharmaceutically acceptable carriers in a solid form such asgranules, tablets, dragees, pellets, troches, capsules, orsuppositories; creams; ointments; aerosols; powders for insufflation; ina liquid form such as solutions, emulsions, or suspensions forinjection; ingestion; eye drops; and any other form suitable for use.And, if necessary, there may be included in the above preparationauxiliary substance such as stabilizing, thickening, wetting,emulsifying and coloring agents; perfumes or buffer; or any othercommonly may be used as additives.

The object polypeptide compound [I] or a pharmaceutically acceptablesalt thereof is/are included in the pharmaceutical composition in anamount sufficient to produce the desired antimicrobial effect upon theprocess or condition of diseases.

For applying the composition to human, it is preferable to apply it byintravenous, intramuscular, pulmonary, oral administration, orinsufflation. While the dosage of therapeutically effective amount ofthe object polypeptide compound [I] varies form and also depends uponthe age and condition of each individual patient to be treated, in thecase of intravenous administration, a daily dose of 0.01-20 mg of theobject polypeptide compound [I] per kg weight of human being in the caseof intramuscular administration, a daily dose of 0.1-20 mg of the objectpolypeptide compound [I] per kg weight of human being, in case of oraladministration, a daily dose of 0.5-50 mg of the object polypeptidecompound [I] per kg weight of human being is generally given fortreating or preventing infectious diseases.

Especially in case of the treatment of prevention of Pneumocystiscarinii infection, the followings are to be noted.

For administration by inhalation, the compounds of the present inventionare conveniently delivered in the form of an aerosol spray presentationfrom pressurized as powders which may be formulated and the powdercompositions may be inhaled with the aid of an insufflation powderinhaler device. The preferred delivery system for inhalation is ametered dose inhalation aerosol, which may be formulated as a suspensionor solution of compound in suitable propellants such as fluorocarbons orhydrocarbons.

Because of desirability to directly treat lung and bronchi, aerosoladministration is a preferred method of administration. Insufflation isalso a desirable method, especially where infection may have spread toears and other body cavities.

Alternatively, parenteral administration may be employed using dripintravenous administration.

The following Preparations and Examples are given for the purpose ofillustrating the present invention in more detail.

Preparation 1

To a solution of 2-amino-4′-bromoacetophenone hydrochloride (8.2 g),4-pentyloxybenzoic acid (6.8 g) and 1-hydroxybenzotriazole (4.42 g) indichloromethane (80 ml) was added1-ethyl-3-(3′-dimethylaminopropyl)carbodiimide (WSCD) (6.0 ml), andstirred for 3 hours at ambient temperature. The reaction mixture wasdiluted with dichloromethane (800 ml), and washed with 1N hydrochloricacid, saturated sodium hydrogen carbonate aqueous solution and brine.The organic layer was dried over magnesium sulfate. Magnesium sulfatewas filtered off, and the filtrate was evaporated under reducedpressure. The solids were slurried in ethyl acetate, and collected byfiltration to give 2-(4-pentyloxybenzamido)-4′-bromoacetophenone (2.49g).

NMR (DMSO-d₆, δ): 0.80-1.00 (3H, m), 1.22-1.55 (4H, m), 1.60-1.85 (2H,m), 4.03 (2H, t, J=6.5 Hz), 4.72 (2H, d, J=5.6 Hz), 7.01 (2H, d, J=8.8Hz), 7.78 (2H, d, J=8.5 Hz), 7.85 (2H, d, J=8.8 Hz), 7.97 (2H, d, J=8.6Hz), 8.72 (1H, t, J=5.6 Hz)

MASS (m/z): 404, 406

Preparation 2

To a solution of 2-(4-pentyloxybenzamido)-4′-bromoacetophenone (3.39 g)in tetrahydrofuran (34 ml) was added phosphorus pentasulfide (2.43 g),and refluxed for 30 minutes. The reaction mixture was cooled, and pouredinto saturated sodium hydrogen carbonate aqueous solution (400 ml), andstirred for 1.5 hours. The resulting precipitate was collected byfiltration to give 2-(4-pentyloxyphenyl)-5-(4-bromophenyl)thiazole (2.32g).

IR (KBr): 2939.0, 2858.0, 1602.6, 1525.4, 1473.3, 1257.4 cm⁻¹

NMR (CDCl₃, δ): 0.80-1.00 (3H, m), 1.25-1.60 (4H, m), 1.70-1.95 (2H, m),4.01 (2H, t, J=6.5 Hz), 6.96 (2H, d, J=8.9 Hz), 7.44 (2H, d, J=8.8 Hz),7.54 (2H, d, J=8.8 Hz), 7.90 (2H, d, J=8.9 Hz), 7.95 (1H, s)

MASS (m/z): 402, 404

Preparation 3

A solution of 2-(4-pentyloxyphenyl)-5-(4-bromophenyl)thiazole (2.3 g) indry tetrahydrofuran (60 ml) was cooled to −60° C., and a solution ofn-butyllithium (1.66M in n-hexane, 4.46 ml) was added slowly to maintainthe reaction temperature at −60° C. After stirring for 1 hour, asolution of n-butyllithium (1.66M in n-hexane, 1.0 ml) was additionallyadded. The reaction mixture was allowed to warm to −40° C. Afterstirring for 30 minutes, dry-ice (5 g) was added. The reaction mixturewas allowed to warm to room temperature over 30 minutes. To the reactionmixture was added water (25 ml) and 0.5N-hydrochloric acid (100 ml),then extracted with dichloromethane (500 ml). The organic layer waswashed with brine, and dried over magnesium sulfate. Magnesium sulfatewas filtered off, and the filtrate was evaporated under reducedpressure. The solids were slurried in acetonitrile, and collected byfiltration to give 4-[2-(4-pentyloxyphenyl)thiazol-5-yl]benzoic acid(1.70 g).

IR (KBr): 2954.4, 2867.6, 2667.1, 2547.5, 1683.6, 1604.5, 1430.9,1295.9, 1253.5 cm⁻¹

NMR (DMSO-d₆, δ): 0.80-1.00 (3H, m), 1.20-1.55 (4H, m), 1.60-1.85 (2H,m), 4.04 (2H, t, J=6.5 Hz), 7.07 (2H, d, J=8.8 Hz), 7.83 (2H, d, J=8.4Hz), 7.91 (2H, d, J=8.8 Hz), 8.01 (2H, d, J=8.4 Hz), 8.39 (1H, s)

MASS (m/z): 368

Preparation 4

To a solution of 1-hydroxybenzotriazole (722 mg) and4-[2-(4-pentyloxyphenyl)thiazol-5-yl]benzoic acid (1.64 g) indichloromethane (33 ml) was added1-ethyl-3-(3′-dimethylaminopropyl)carbodiimide hydrochloride (WSC.HCl)(1.28 g), and stirred for 2 hours at ambient temperature. To thereaction mixture was added dichloromethane (500 ml) to be clearsolution, then washed with water (200 ml×2) and brine, and dried overmagnesium sulfate. Magnesium sulfate was filtered off, and the filtratewas evaporated under reduced pressure. The solids were slurried inacetonitrile, and collected by filtration to give4-[2-(4-pentyloxyphenyl)-thiazol-5-yl]benzoic acid benzotriazol-1-ylester (1.27 g).

IR (KBr): 3461.6, 2948.6, 2869.6, 1781.9, 1602.6, 1261.2, 985.4 cm⁻¹

NMR (CDCl₃, δ): 0.90-1.05 (3H, m), 1.30-1.60 (4H, m), 1.70-1.95 (2H, m),4.03 (2H, t, J=6.5 Hz), 6.99 (2H, d, J=8.9 Hz), 7.40-7.70 (3H, m), 7.82(2H, d, J=8.6 Hz), 7.94 (2H, d, J=8.9 Hz), 8.12 (2H, d, J=8.1 Hz), 8.17(1H, s), 8.31 (2H, d, J=8.6 Hz)

MASS (m/z): 485

The following compound was obtained according to a similar manner tothat of Preparation 1.

Preparation 5

2-(4-Hexyloxybenzamido)-4′-bromoacetophenone

IR (KBr): 3317.0, 2937.1, 2867.6, 1699.0, 1637.3, 1556.3, 1508.1, 1253.5cm⁻¹

NMR (DMSO-d₆, δ): 0.80-1.00 (3H, m), 1.20-1.55 (6H, m), 1.60-1.85 (2H,m), 4.03 (2H, t, J=6.4 Hz), 4.72 (2H, d, J=5.5 Hz), 7.01 (2H, d, J=8.8Hz), 7.77 (2H, d, J=8.6 Hz), 7.85 (2H, d, J=8.8 Hz), 7.97 (2H, d, J=8.6Hz), 8.72 (1H, t, J=5.6 Hz)

MASS (m/z): 418, 420

The following compound was obtained according to a similar manner tothat of Preparation 2.

Preparation 6

2-(4-Hexyloxyphenyl)-5-(4-bromophenyl)thiazole

IR (KBr): 2937.1, 2854.1, 1602.6, 1523.5, 1475.3, 1438.6, 1255.4, 833.1cm⁻¹

NMR (CDCl₃, δ): 0.80-1.10 (3H, m), 1.20-1.60 (6H, m), 1.70-1.95 (2H, m),4.01 (2H, t, J=6.5 Hz), 6.96 (2H, d, J=8.9 Hz), 7.44 (2H, d, J=8.7 Hz),7.54 (2H, d, J=8.7 Hz), 7.89 (2H, d, J=8.9 Hz), 7.94 (1H, s)

MASS (m/z): 416, 418

The following compound was obtained according to a similar manner tothat of Preparation 3.

Preparation 7

4-[2-(4-Hexyloxyphenyl)thiazol-5-yl]benzoic acid

IR (KBr): 2933.2, 2863.8, 2669.0, 2547.5, 1679.7, 1604.5, 1513.8,1432.9, 1297.9, 1251.6 cm⁻¹

NMR (DMSO-d₆, δ): 0.80-1.00 (3H, m), 1.15-1.60 (6H, m), 1.60-1.85 (2H,m), 4.05 (2H, t, J=6.5 Hz), 7.07 (2H, d, J=8.8 Hz), 7.83 (2H, d, J=8.4Hz), 7.91 (2H, d, J=8.8 Hz), 8.01 (2H, d, J=8.4 Hz), 8.40 (1H, S)

MASS (m/z): 382

The following compounds [Preparation 8 to 10] were obtained according toa similar manner to that of Preparation 4.

Preparation 8

4-[2-(4-Hexyloxyphenyl)thiazol-5-yl]benzoic acid benzotriazol-1-yl ester

IR (KBr): 2927.4, 2865.7, 1774.2, 1602.6, 1434.8, 1251.6, 1187.9, 993.2cm⁻¹

NMR (CDCl₃, δ): 0.85-1.05 (3H, m), 1.20-1.60 (6H, m), 1.60-1.95 (2H, m),4.03 (2H, t, J=6.5 Hz), 6.99 (2H, d, J=8.8 Hz), 7.35-7.70 (3H, m), 7.81(2H, d, J=8.5 Hz), 7.94 (2H, d, J=8.8 Hz), 8.12 (2H, d, J=8.1 Hz), 8.17(1H, s), 8.31 (2H, d, J=8.5 Hz)

MASS (m/z): 499

Preparation 9

4-[5-(4-Hexyloxyphenyl)thiazol-2-yl]benzoic acid benzotriazol-1-yl ester

IR (KBr): 2946.7, 2865.7, 1776.1, 1604.5, 1251.6, 1230.4 cm⁻¹

NMR (CDCl₃, δ): 0.80-1.05 (3H, m), 1.20-1.65 (6H, m), 1.70-1.95 (2H, m),4.01 (2H, t, J=6.5 Hz), 6.97 (2H, d, J=8.8 Hz), 7.40-7.65 (5H, m), 8.03(1H, s), 8.12 (1H, d, J=8.2 Hz), 8.18 (2H, d, J=8.7 Hz), 8.36 (2H, d,J=8.7 Hz)

MASS (m/z): 499

Preparation 10

4-[5-[4-(4-Propoxyphenyl)phenyl]thiazol-2-yl]benzoic acidbenzotriazol-1-yl ester

IR (KBr): 2967.9, 2937.1, 2875.3, 1772.3, 1600.6, 1249.6 cm⁻¹

NMR (CDCl₃, δ): 1.07 (3H, t, J=7.4 Hz), 1.75-1.95 (2H, m), 3.99 (2H, t,J=6.6 Hz), 7.01 (2H, d, J=8.7 Hz), 7.40-7.60 (3H, m), 7.60 (2H, d, J=8.7Hz), 7.69 (2H, d, J=8.4 Hz), 7.85 (2H, d, J=8.5 Hz), 8.06 (2H, d, J=8.4Hz), 8.13 (2H, d, J=8.1 Hz), 8.24 (1H, s), 8.34 (2H, d, J=8.5 Hz)

MASS (m/z): 533

Preparation 11

To a solution of 2-amino-4′-methoxyacetophenone hydrochloride (5.33 g)in pyridine (4.28 ml), triethylamine (3.68 ml) and dichloromethane (50ml) was added dropwise a solution of terephthalic acid monomethyl ester(5.25 g) in dichloromethane (10 ml) at 0° C. The reaction mixture wasallowed to warm to room temperature, and stirred for 3 hours. To thereaction mixture was added dichloromethane (200 ml), washed with1N-sodium hydroxide (100 ml×2), saturated hydrogen carbonate aqueoussolution (100 ml×2) and brine, and dried over magnesium sulfate.Magnesium sulfate was filtered off, and the filtrate was evaporatedunder reduced pressure. The solids were slurried in n-hexane, andcollected by filtration to give2-(4-methoxycarbonyl-benzamido)-4′-methoxyacetophenone (7.97 g).

IR (KBr): 3401.8, 3378.7, 2956.3, 2844.5, 1724.0, 1683.6, 1643.1,1602.6, 1290.1, 1263.1 cm⁻¹

NMR (CDCl₃, δ): 3.91 (3H, s), 3.96 (3H, s), 4.91 (2H, d, J=4.1 Hz), 7.00(2H, d, J=8.9 Hz), 7.40 (1H, t, J=4.1 Hz), 7.94 (2H, d, J=8.6 Hz), 8.02(2H, d, J=8.9 Hz), 8.14 (2H, d, J=8.6 Hz)

MASS (m/z): 328

Preparation 12

To a solution of 2-(4-methoxycarbonylbenzamido)-4′-methoxyacetophenone(4.0 g) in tetrahydrofuran (80 ml) was added phosphorus pentasulfide(3.53 g), and refluxed for 2.5 hours. The reaction mixture wasadditionally added phosphorus pentasulfide (1.0 g), and refluxed for 1hour. The reaction mixture was filtered, and the filtrate was cooled,and poured into water (1200 ml), and stirred for 1 hour. The resultingprecipitate was collected by filtration. The solids were slurried inacetonitrile, and collected by filtration to give methyl4-[5-(4-methoxyphenyl)thiazol-2-yl]benzoate (3.26 g).

IR (KBr): 2950.6, 2840.6, 1712.5, 1604.5, 1280.5, 1249.6 cm⁻¹

NMR (CDCl₃, δ): 3.86 (3H, s), 3.95 (3H, s), 6.96 (2H, d, J=8.8 Hz), 7.55(2H, d, J=8.8 Hz), 7.97 (1H, s), 8.02 (2H, d, J=8.7 Hz), 8.12 (2H, d,J=8.7 Hz)

MASS (m/z): 326

Preparation 13

To a solution of boron tribromide (1.0M in dichloromethane, 97.7 ml) wasadded dropwise methyl 4-[5-(4-methoxyphenyl)thiazol-2-yl]benzoate (3.18g) in dichloromethane (50 ml) at −78° C. The reaction mixture wasallowed to warm to room temperature, and stirred for 2 hours. Thereaction mixture was poured into ice-water (1000 ml), and stirred for 30minutes at room temperature. The precipitate was collected byfiltration, washed with water, and dried overnight to give in theproportion of 37:63 mixture of methyl4-[5-(4-hydroxyphenyl)thiazol-2-yl]benzoate and4-[5-(4-hydroxyphenyl)thiazol-2-yl]benzoic acid (3.07 g), and those wereused without purification in the next reaction.

Preparation 14

To a suspension of a mixture of methyl4-[5-(4-hydroxyphenyl)thiazol-2-yl]benzoate and4-[5-(4-hydroxyphenyl)thiazol-2-yl]benzoic acid (1.0 g), potassiumcarbonate (930 mg) in N,N-dimethylformamide (5 ml) was addedn-hexylbromide (0.95 ml), and stirred at 100° C. (bath temperature).After 3.5 hours, to the reaction mixture was additionally addedn-hexylbromide (0.25 ml), and stirred at 100° C. (bath temperature) for1 hour. After cooling, the mixture was added to 0.1N hydrochloric acid(100 ml). The resulting precipitate was collected by filtration, washedwith water, and dried overnight. To this material was added the solutionof tetrahydrofuran (20 ml), methanol (20 ml) and 10% sodium hydroxideaqueous solution (2.5 ml). The mixture was refluxed for 2 hours. Aftercooling, the reaction mixture was diluted with water (100 ml), andadjusted to pH 2 with 1N hydrochloric acid. The resulting precipitatewas collected by filtration. The solid was collected by filtration,washed with water and acetonitrile, and dried to give4-[5-(4-hexyloxyphenyl)thiazol-2-yl]benzoic acid (621 mg).

IR (KBr): 2933.2, 2865.7, 2661.3, 2539.8, 1681.6, 1604.5, 1430.9,1288.2, 1251.6 cm⁻¹

NMR (CDCl₃, δ): 1.80-1.00 (3H, m), 1.20-1.60 (6H, m), 1.60-1.85 (2H, m),4.02 (2H, t, J=6.5 Hz), 7.04 (2H, d, J=8.8 Hz), 7.67 (2H, d, J=8.8 Hz),8.06 (4H, s), 8.28 (1H, s), 13.20 (1H, br s)

MASS (m/z): 382

Preparation 15

To a solution of 4′-ethoxycarbonyl-2-bromoacetophenone (2.0 g) inN,N-dimethylformamide (8 ml) was added sodium azide (480 mg) at 0° C.,and stirred for 2 hours at room temperature. The reaction mixture waspoured into water (100 ml), extracted with diethyl ether (200 ml×2),washed with brine, and dried over magnesium sulfate. Magnesium sulfatewas filtered off, and the filtrate was evaporated under reducedpressure. The product was purified by silica gel column chromatography(ethyl acetate:hexane=1:5) to give 2-azido-4′-ethoxycarbonylacetophenone(1.46 g).

IR (KBr): 2991.1, 2904.3, 2104.0, 1702.8, 1288.2 cm⁻¹

NMR (CDCl₃, δ): 1.42 (3H, t, J=7.1 Hz), 4.42 (2H, q, J=7.1 Hz), 4.60(1H, s), 7.96 (2H, d, J=8.7 Hz), 8.17 (2H, d, J=8.7 Hz)

MASS (m/z): 206

Preparation 16

To a solution of 2-azido-4′-ethoxycarbonylactophenone (1.44 g), methanol(41.3 ml) and conc. hydrochloric acid (1.38 ml) was added 10%palladium-activated carbon (73 mg), and stirred for 3 hours in hydrogenatmosphere at 2.8 atm. After 1 hour, the reaction mixture was filteredoff, and the filtrate was evaporated under reduced pressure. The solidswere slurried in diisopropyl ether, and collected by filtration to give2-amino-4′-ethoxycarbonylacetophenone hydrochloride (1.40 g).

NMR (CDCl₃, δ): 1.35 (3H, t, J=7.1 Hz), 4.37 (2H, q, J=7.1 Hz), 4.65(2H, s), 8.12 (2H, d, J=9.0 Hz), 8.17 (2H, d, J=9.0 Hz)

MASS (m/z): 208

Preparation 17

To a solution of 2-amino-4′-ethoxycarbonylacetophenone hydrochloride(700 mg), 4-(4-propoxyphenyl)benzoic acid (866 mg) and1-hydroxybenzotriazole (456.5 mg) in triethylamine (0.47 ml) anddichloromethane (7 ml) was added1-ethyl-3-(3′-dimethylaminopropyl)carbodiimide hydrochloride (WSC.HCl)(648 mg), and stirred for 5 hours at ambient temperature. To thereaction mixture was added water (100 ml), and extracted withdichloromethane. The organic layer was washed with 1N hydrochloric acid,water, saturated sodium hydrogen carbonate aqueous solution and brine.Then the organic layer was dried over magnesium sulfate. Magnesiumsulfate was filtered off, and the filtrate was evaporated under reducedpressure. The solids were slurried in ethyl acetate, and collected byfiltration to give2-[4-(4-propoxyphenyl)benzoylamino]-4′-ethoxycarbonylacetophenone (746mg).

IR (KBr): 3320.8, 2966.0, 2931.3, 2875.3, 1718.3, 1699.0, 1645.0,1606.4, 1280.5 cm⁻¹

NMR (DMSO-d₆, δ): 1.00 (3H, t, J=7.4 Hz), 1.35 (3H, t, J=7.1 Hz), 1.76(2H, q, J=7.3 Hz), 3.99 (2H, t, J=6.5 Hz), 4.36 (2H, q, J=7.1 Hz), 4.82(1H, d, J=5.6 Hz), 7.05 (2H, d, J=8.8 Hz), 7.69 (2H, d, J=8.8 Hz), 7.76(2H, d, J=8.4 Hz), 7.96 (2H, d, J=8.4 Hz), 8.11 (2H, d, J=8.7 Hz), 8.18(2H, d, J=8.7 Hz), 8.97 (1H, t, J=5.6 Hz)

MASS (m/z): 446

Preparation 18

To a solution of2-[4-(4-propoxyphenyl)benzoylamino]-4′-ethoxycarbonylacetophenone (709.4mg) in tetrahydrofuran (7 ml) was added phosphorus pentasulfide (460mg), and refluxed for 2 hours and 45 minutes. The reaction mixture waspoured into ice-water (500 ml), neutralized by saturated sodium hydrogencarbonate aqueous solution, and stirred for 1 hour. The resultingprecipitate was collected by filtration. The solids were slurried inacetonitrile, and collected by filtration to give ethyl4-[2-(4′-propoxy-4-biphenyl)thiazol-5-yl]benzoate (481 mg).

IR (KBr): 2967.9, 2875.3, 1710.6, 1604.5, 1280.5 cm⁻¹

MASS (m/z): 444

Preparation 19

To a solution of ethyl4-[2-(4′-propoxy-4-biphenyl)-thiazol-5-yl]benzoate (461.4 mg) indichloromethane (20 ml), tetrahydrofuran (4 ml) and ethanol (200 ml) wasadded 10% sodium hydroxide aqueous solution (6.1 ml), and refluxed for30 minutes. To the reaction mixture was added water (100 ml), andrefluxed for 10 minutes. After cooling, the reaction mixture wasadjusted to pH 2 with 1N hydrochloric acid. The resulting precipitatewas collected by filtration, and washed with water. The solids wereslurried in acetonitrile, and collected by filtration to give4-[2-(4′-propoxy-4-biphenyl)thiazol-5-yl]benzoic acid (350 mg).

IR (KBr): 3455.8, 2966.0, 2877.3, 2676.7, 2551.4, 1685.5, 1604.5,1432.9, 1286.3, 1253.5 cm⁻¹

NMR (DMSO-d₆, δ): 1.00 (3H, t, J=7.4 Hz), 1.65-1.90 (2H, m), 3.99 (2H,t, J=6.5 Hz), 7.06 (2H, d, J=8.8 Hz), 7.76 (2H, d, J=8.5 Hz), 7.87 (2H,d, J=8.5 Hz), 8.02 (2H, d, J=8.5 Hz), 8.04 (2H, d, J=8.5 Hz), 8.49 (1H,s)

MASS (m/z): 416

EXAMPLE 1

(1) Fermentation

The seed medium (30 ml) consisting of sucrose 4%, glucose 1%, solublestarch 2%, cotton seed flour 3%, soybean powder 1.5%, KH₂PO₄ 1%, CaCO₃0.2%, Adekanol LG-109 (Asahi Denka Co., Ltd.) 0.05% and Silicone KM-70(Shin-Etsu Chemical Co., Ltd.) 0.05% was poured into a 100 ml-Erlenmeyerflask and sterilized at 121° C. for 30 minutes. A loopful of slantculture of Coleophoma crateriformis No. 738 was inoculated to the mediumand cultured at 25° C. for 5 days on a rotary shaker.

Four 8 ml portions of the seed culture were transferred to four 500ml-Erlenmeyer flasks each containing 160 ml of the same seed medium, andcultured at 25° C. for 2 days on a rotary shaker (220 rpm, 5.1cm-throw).

The resultant second seed culture was inoculated to a medium (20 l)consisting of modified starch 3%, starch acid hydrolysates 6%, cottonseed flour 1%, chicken meat bone meal 1%, dried yeast 2%, (NH₄)₂SO₄0.05%, NaH₂PO₄.12H₂O 0.5%, β-cyclodextrin 1%, Adekanol LG-109 0.05% andSilicone KM-70 0.05% in a 30 l-jar fermentor, which had been sterilizedat 121° C. for 30 minutes in advance, and cultured at 25° C. for 4 daysunder aeration of 20 l/min. (internal pressure: 1 kg/cm²) and agitationof 250 rpm.

The production of active compound in the fermentation broth wasmonitored by HPLC analysis.

(2) Isolation and Purification

After the culture was completed, an equal volume of acetone was added tothe cultured broth (20 l). The mixture was allowed to stand for about anhour with stirring at room temperature. The resultant mixture wasfiltered with an aid of diatomaceous earth, yielding about 35 l of thefiltrate. The filtrate was concentrated in vacuo to an aqueous solutionand passed through a column (1 l) of Diaion HP-20 (Mitsubishi ChemicalCo., Ltd.) packed with water. The column was washed with water (3 l) and50% aqueous methanol (3 l) and then eluted with methanol (3 l). Theeluate was concentrated in vacuo to give residual water. This residuewas extracted twice with an equal volume of ethyl acetate. The aqueouslayer was passed through a column (100 ml) of YMC GEL (ODS-AM 120-S50,YMC Co., Ltd.) packed with water. The column was washed with 20% aqueousacetonitrile containing 0.5% NaH₂PO₄.2H₂O (300 ml) and 40% aqueousacetonitrile containing 0.5% NaH₂PO₄.2H₂O (300 ml) and then eluted with50% aqueous acetonitrile containing 0.5% NaH₂PO₄.2H₂O (200 ml). Theeluate was concentrated in vacuo to an aqueous solution. This residuewas passed through a column (20 ml) of Diaion HP-20 packed with water.The column was washed with water (200 ml) and then eluted with 80%aqueous methanol (60 ml). The eluate was concentrated in vacuo to anaqueous solution. This residue was applied onto a column (175 ml) or YMCGEL ODS-AM 120-S50 packed with water, and the column was eluted with 45%aqueous acetonitrile containing 0.5% NaH₂PO₄.2H₂O. Fractions containingthe WF 738A and the WF 738C were combined and concentrated in vacuorespectively. The WF 738A and the WF 738C were finally purified bypreparative HPLC, using a YMC-packed column (ODS-AM SH-343 5AM S-5 (YMCCo., Ltd., 250 mm L.×20 mm I.D.), with 55% aqueous acetonitrilecontaining 0.5% NaH₂PO₄.2H₂O as a mobile phase and flow rate of 9.9ml/min.).

The portion corresponding to the WF 738A was concentrated in vacuo togive residual water. This residue was passed through a column (10 ml) ofDiaion HP-20 packed with water. The column was washed with water (100ml) and eluted with 80% aqueous methanol (30 ml). The eluate wasconcentrated and lyophilized to give 22 mg of the WF 738A as a whitepowder.

The portion corresponding to the WF 738C was concentrated in vacuo togive residual water. This residue was passed through a column (2 ml) ofDiaion HP-20 packed with water. The column was washed with water (20 ml)and eluted with 80% aqueous methanol (6 ml). The eluate was concentratedand lyophilized to give 4 mg of the WF 738C as a white powder.

The WF 738A as obtained has the following physico-chemical properties.

HPLC Condition

Column: YMC Pack ODS-AM303 (250 mm L.×4.6 mm I.D.; YMC Co., Ltd.)

Eluent: 55% acetonitrile−0.5% NaH₂PO₄.2H₂O

Flow rate: 1 ml/min

Detection: UV at 210 nm

Retention time: 8.1 minutes

Appearance

white powder

Nature

acid substance

Molecular Formula

C₅₀H₇₉N₈O₂₀SNa [free acid: C₅₀H₈₀N₈O₂₀S]

Molecular Weight

Molecular weight: 1144 (free acid)

ESI-MS: (m/z) 1145 (M+H)⁺

Elementary Analysis

Calcd. for C₅₀H₇₉N₈O₂₀SNa.6H₂O C, 47.09; H, 7.19; N, 8.79; S, 2.51; (%)

Found: C, 47.23, H, 7.35; N, 8.56; S, 2.18; (%)

Melting Point

173-177° C. (dec.)

Specific Rotation

[α]_(D) ²³−10° (C 0.5, methanol)

Ultraviolet Absorption Spectrum

λ_(max) ^(methanol): 278 nm (ε1487)

Solubility

Soluble: water, methanol, dimethylsulfoxide

Insoluble: chloroform

Color Reaction

Positive: iodine vapor reaction, ceric sulfate reaction

Negative: ninhydrin reaction, Molish reaction, Dragendorff reaction,ferric chloride reaction

Thin Layer Chromatography (TLC)

Stationary phase Developing solvent Rf value Silica Gel 60 F₂₅₄n-butanol:acetic acid:water 0.41 (E. Merck) (4:1:2)

Infrared Spectrum

ν_(max) ^(KBr): 3350, 2930, 2850, 1650, 1640, 1520, 1460, 1270, 1250,1050 (cm⁻¹)

¹H-NMR (500 MHz, CD₃OD, δ): 7.17 (1H, d, J=2 Hz), 6.89 (1H, dd, J=8 and2 Hz), 6.81 (1H, d, J=8 Hz), 5.27 (1H, d, J=3 Hz), 5.08 (1H, d, J=4 Hz),4.92 (1H, m), 4.63 (1H, dd, J=11 and 7 Hz), 4.57 (1H, m), 4.49 (1H, brs), 4.43 (1H, m), 4.41-4.36 (3H, m), 4.23 (1H, dd, J=12 and 4 Hz), 4.18(1H, m), 4.09 (1H, m), 3.97 (1H, dd, J=11 and 3 Hz), 3.91 (1H, m), 3.78(1H, br d, J=11 Hz), 3.74 (1H, br d, J=12 Hz), 3.36 (1H, m), 2.62 (1H,dd, J=16 and 4.5 Hz), 2.59 (2H, m), d, J=7 Hz), 2.53 (1H, m), 2.47 (1H,m), 2.43 (1H, dd, J=16 and 9 Hz), 2.21 (2H, m), 2.07-1.99 (2H, m), 1.94(1H, m), 1.58 (2H, m), 1.34-1.23 (24H, m), 1.07 (3H, d, J=7 Hz), 0.89(3H, t, J=7 Hz)

¹³C-NMR (125 MHz, D₂O, δ): 176.7 (s), 176.2 (s), 174.2 (s), 173.8 (s),172.6 (s), 172.4 (s), 172.0 (s), 169.5 (s), 149.1 (s), 141.1 (s), 131.0(s), 128.0 (d), 125.3 (d), 118.2 (d), 75.6 (d), 74.3 (d), 73.8 (d), 71.3(d), 70.7 (d), 70.6 (d), 70.1 (d), 63.6 (t), 62.4 (d), 58.4 (d), 57.0(t), 56.0 (d), 55.4 (d), 52.9 (t), 51.3 (d), 40.8 (t), 39.6 (t), 39.1(d), 39.0 (t), 36.7 (t), 35.5 (t), 33.1 (t), 30.8 (t)×5, 30.7 (t), 30.6(t), 30.5 (t), 30.4 (t), 30.3 (t), 26.9 (t), 23.7 (t), 14.4 (q), 11.1(q)

From the analysis of the above physical and chemical properties, and theresult of the further investigation of identification of chemicalstructure, the chemical structure of the WF 738A has been identified andassigned as follows.

The WF 738C as obtained has the following physico-chemical properties.

HPLC Condition

Column: YMC Pack ODS-AM303 (250 mm L.×4.6 mm I.D.; YMC Co., Ltd.)

Eluent: 55% acetonitrile−0.5% NaH₂PO₄.2H₂O

Flow rate: 1 ml/min

Detection: UV at 210 nm

Retention time: 9.0 minutes

Appearance

white powder

Nature

acid substance

Molecular Formula

C₅₀H₇₉N₈O₁₉SNa [free acid: C₅₀H₈₀N₈O₁₉S]

Molecular Weight

Molecular weight: 1128 (free acid)

ESI-MS: (m/z) 1129 (M+H)⁺

Melting Point

155-160° C. (dec.)

Specific Rotation

[α]_(D) 23 −2° (C 0.35, methanol)

Ultraviolet Absorption Spectrum

λ_(max) methanol: 277 nm (ε 1579)

Solubility

Soluble: water, methanol, dimethylsulfoxide

Insoluble: chloroform

Color Reaction

Positive: iodine vapor reaction, ceric sulfate reaction

Negative: ninhydrin reaction, Molish reaction, Dragendorff reaction,ferric chloride reaction

Thin Layer Chromatography (TLC)

Stationary phase Developing solvent Rf value Silica Gel 60 F₂₅₄n-butanol:acetic acid:water 0.43 (E. Merck) (4:1:2)

Infrared Spectrum

ν_(max) KBr: 3350, 2920, 2850, 1650, 1640, 1540, 1520, 1450, 1270, 1050(cm⁻¹)

¹H-NMR (500 MHz, CD₃OD, δ): 7.17 (1H, d, J=2 Hz), 6.89 (1H, dd, J=8 and2 Hz), 6.80 (1H, d, J=8 Hz), 5.38 (1H, dd, J=10.5 and 3.5 Hz), 5.10 (1H,d, J=4 Hz), 4.94 (1H, br d, J=4 Hz), 4.60 (1H, dd, J=11 and 7 Hz), 4.57(1H, m), 4.54 (1H, d, J=2 Hz), 4.42 (1H, m), 4.39-4.30 (3H, m), 4.26(1H, dd, J=12 and 4 Hz), 4.13-4.07 (2H, m), 3.97 (1H, dd, J=11 and 4Hz), 3.77-3.71 (2H, m), 3.35 (1H, m), 2.65 (1H, dd, J=16 and 4 Hz),2.62-2.52 (3H, m), 2.46 (1H, m), 2.42 (1H, dd, J=16 and 9 Hz), 2.25 (2H,m), 2.08-1.96 (3H, m), 1.74-1.64 (2H, m), 1.59 (2H, m), 1.34-1.24 (24H,m), 1.07 (3H, d, J=7 Hz), 0.89 (3H, t, J=7 Hz)

¹³C-NMR (125 MHz, CD₃OD, δ): 176.9 (s), 176.3 (s), 174.5 (s), 174.1 (s),172.8 (s), 172.5 (s), 171.8 (s), 169.1 (s), 149.1 (s), 141.1 (s), 131.1(s), 128.0 (d), 125.3 (d), 118.2 (d), 76.0 (d), 73.9 (d), 71.9 (d), 71.3(d), 70.6 (d), 70.3 (d), 63.8 (t), 62.5 (d), 58.2 (d), 57.0 (t), 56.0(d), 55.4 (d), 52.9 (t), 52.0 (d), 40.8 (t), 39.5 (t), 39.1 (d), 39.0(t), 36.8 (t), 33.1 (t), 30.9 (t), 30.8 (t)×5, 30.7 (t), 30.6 (t), 30.5(t), 30.4 (t), 30.3 (t), 27.3 (t), 27.0 (t), 23.7 (t), 14.4 (q), 11.1(q)

From the analysis of the above physical and chemical properties, and theresult of the further investigation of identification of chemicalstructure, the chemical structure of the WF 738C has been identified andassigned as follows.

EXAMPLE 2

(1) Fermentation

The fermentation of Example 2 was carried out according to a similarmanner to that of Example 1.

(2) Isolation and Purification

After the culture was completed, an equal volume of acetone was added tothe cultured broth (20 l). The mixture was allowed to stand for about anhour with stirring at room temperature. The resultant mixture wasfiltered with an aid of diatomaceous earth, yielding about 30 l of thefiltrate. The filtrate was diluted with an equal volume of water andpassed through a column (1l ) of Diaion HP-20 (Mitsubishi Chemical Co.,Ltd.) packed with water. The column was washed with water (3 l) and 50%aqueous methanol (3 l) and then eluted with 80% aqueous methanol (3 l).The eluate was diluted with an equal volume of water. and applied onto acolumn (1 l) of YMC GEL (ODS-AM 120-S50, YMC Co., Ltd.) packed withwater, and the col was eluted with 45% aqueous acetonitrile containing0.5% NaH₂PO₄.2H₂O. Fractions containing the WF 738B were combined andconcentrated in vacuo. The WF 738B was further purified by preparativeHPLC, using a YMC-packed column (ODS-AM SH-343 5AM S-5, YMC Co., Ltd.,250 mm L.×20 mm I.D.) with 40% aqueous acetonitrile containing 0.3%NaH₂PO₄.2H₂O as a mobile phase and flow rate of 9.9 ml/min.

Fractions containing the WF 738B were collected and concentrated invacuo to give residual water. This residue was passed through a column(10 ml) of Diaion HP-20 packed with water. The column was washed withwater (100 ml) and eluted with 80% aqueous methanol (30 ml). The eluatewas concentrated in vacuo and lyophilized to give a white powder.

The powder was dissolved in methanol and preabsorbed on a small amountof silica gel and applied onto a silica gel column (20 ml) prepared withacetone. The column was eluted stepwise with acetone-methanol (10:1,5:1, 3:1 and 1:1 v/v). The eluate from acetone-methanol (3:1 v/v) wasconcentrated in vacuo to give a white powder. The powder was dissolvedin water and applied onto a column (1 ml) of Diaion HP-20 packed withwater. The column was washed with water (5 ml) and eluted with 80%aqueous methanol (3 ml). The eluate was concentrated in vacuo andlyophilized to give 2.4 mg of the WF 738B as a white powder.

The WF 738B as obtained has the following physico-chemical properties.

HPLC Condition

Column: YMC Pack ODS-AM AM303, S-5 120A (250 mm L.×4.6 mm I.D.; YMC Co.,Ltd.)

Eluent: 50% acetonitrile—0.5% NaH₂PO₄.2H₂O

Flow rate: 1 ml/min

Detection: UV at 210 nm

Retention time: 10.3 minutes

Appearance

white powder

Molecular Formula

C₄₉H₇₇N₈O₂₀SNa [free acid: C₄₉H₇₈N₈O₂₀S]

Molecular Weight

Molecular weight: 1130 (free acid)

ESI-MS (negative): (m/z) 1129 (M−H)⁻

ESI-MS (positive): (m/z) 1131 (M+H)⁺

Melting Point

160-164° C. (dec.)

Specific Rotation

[α]_(D) 23−7.6° (C 0.5, methanol)

Ultraviolet Absorption Spectrum

λ_(max) water: 275 nm (ε 1900)

Solubility

Soluble: water, methanol

Insoluble: n-hexane

Color Reaction

Positive: iodine vapor reaction, ceric sulfate reaction, ninhydrinreaction

Negative: Molish reaction, ferric chloride reaction

Thin Layer Chromatography (TLC)

Stationary phase Developing solvent Rf value Silica Gel 60 F₂₅₄n-butanol:acetic acid:water 0.45 (E. Merck) (4:1:2)

Infrared Spectrum

ν_(max) KBr: 3360, 2930, 2850, 1650 1630, 1540, 1520, 1440, 1280, 1250,1050 (cm⁻¹)

¹H-NMR (500 MHz, D₂O, δ): 7.17 (1H, d, J=2 Hz), 6.89 (1H, dd, J=8 and 2Hz), 6.80 (1H, d, J=8 Hz), 5.28 (1H, d, J=3 Hz), 5.11 (1H, d, J=4 Hz),4.92 (1H, m), 4.63 (1H, m), 4.57 (1H, m), 4.49 (1H, m), 4.46-4.35 (4H,m), 4.30 (1H, m), 4.23 (1H, m), 4.01-3.89 (3H, m), 3.83-3.72 (3H, m),2.64-2.57 (3H, m) 2.48-2.38 (2H, m), 2.31-2.19 (3H, m), 2.09-1.88 (4H,m), 1.58 (2H, m), 1.34-1.23 (24H, m), 0.89 (3H, t, J=7 Hz)

¹³C-NMR (125 MHz, CD₃OD, δ): 176.7 (s), 176.2 (s), 174.2 (s), 173.8 (s),172.7 (s), 172.6 (s), 172.0 (s), 169.5 (s), 149.1 (s), 141.1 (s), 131.0(s), 128.0 (d), 125.3 (d), 118.2 (d), 74.2 (d), 73.9 (d), 73.8 (d), 71.3(d), 70.7 (d), 70.6 (d), 69.7 (d), 63.6 (t), 62.4 (d), 58.3 (d), 57.0(t), 56.0 (d), 55.4 (d), 51.2 (d), 46.9 (t), 40.8 (t), 39.5 (t), 39.0(t), 36.8 (t), 35.5 (t), 34.6 (t), 33.1 (t), 30.8 (t) ×5, 30.7 (t), 30.6(t), 30.5 (t), 30.4 (t), 30.3 (t), 26.9 (t), 23.7 (t), 14.4 (q)

From the analysis of the above physical and chemical properties, and theresult of the further investigation of identification of chemicalstructure, the chemical structure of the WF 738B has been identified andassigned as follows.

EXAMPLE 3

(1) Fermentation

The fermentation of Example 3 was carried out according to a similarmanner to that of Example 1.

(2) Isolation and Purification

After the culture was completed, an equal volume of acetone was added tothe cultured broth (20 l). The mixture was allowed to stand for about anhour with stirring at room temperature. The resultant mixture wasfiltered with an aid of diatomaceous earth, yielding about 30 l of thefiltrate. The filtrate was diluted with an equal volume of water andpassed through a column (1 l) of Diaion HP-20 (Mitsubishi Chemical Co.,Ltd.) packed with water. The column was washed with water (3 l) and 50%aqueous methanol (3 l) and then eluted with 80% aqueous methanol (3 l).The eluate was diluted with an equal volume of water and applied onto acolumn (1 l) of YMC GEL (ODS-AM 120-S50, YMC Co., Ltd.) packed withwater, and the column was eluted with 45% aqueous acetonitrilecontaining 0.5% NaH₂PO₄.2H₂O. Fractions containing the WF 738D2 werecombined and concentrated in vacuo. The WF 738D2 was further purified bypreparative HPLC, using a YMC-packed column (ODS-AM SH-343 5AM S-5, YMCCo., Ltd., 250 mm L.×20 mm I.D.) with 40% aqueous acetonitrilecontaining 0.3% NaH₂PO₄.2H₂O as a mobile phase and flow rate of 9.9ml/min.

Fractions containing the WF 738D2 were collected and concentrated invacuo to give residual water. This residue was passed through a column(15 ml) of Diaion HP-20 packed with water. The column was washed withwater (150 ml) and eluted with 80% aqueous methanol (50 ml). The eluatewas concentrated in vacuo and lyophilized to give a white powder.

The powder was dissolved in methanol and preabsorbed on a small amountof silica gel and applied onto a column (10 ml) of silica gel 60(230-400 mesH, Merck) prepared with acetone. The column was eluted withacetone-methanol (10:1 v/v). Fractions containing the FW 738D2 werecombined and concentrated in vacuo. The WF 738D2 was further purified bypreparative HPLC, using a YMC-packed column (ODS-AM SH-343 5AM S-5, YMCCo., Ltd., 250 mm L.×20 mm I.D.) with 50% aqueous acetonitrilecontaining 0.5% NaH₂PO₄.2H₂O as a mobile phase and flow rate of 9.9ml/min. Fractions containing the WF 738D2 were collected andconcentrated in vacuo to give residual water. This residue was passedthrough a column (3 ml) of Diaion HP-20 packed with water. The columnwas washed with water (15 ml) and eluted with 80% aqueous methanol (12ml). The eluate was added 1-butanol and concentrated in vacuo andlyophilized to give 2.8 mg of the WF 738D2 as a white powder.

The WF 738D2 as obtained has the following physico-chemical properties.

HPLC Condition

Column: YMC Pack ODS-AM303 (250 mm L.×4.6 mm I.D.; YMC Co., Ltd.)

Eluent: 50% acetonitrile—0.5% NaH₂PO₄.2H₂O.

Flow rate: 1 ml/min

Detection: UV at 210 nm

Retention time: 13.1 minutes

Appearance:

white powder

Molecular Formula

C₅₀H₇₉N₈O₁₈SNa [free acid: C₅₀H₈₀N₈O₁₈S]

Molecular Weight

Molecular weight: 1112 (free acid)

ESI-MS (negative): (m/z) 1111 (M−H)⁻

ESI-MS (positive): (m/z) 1113 (M+H)⁺

Specific Rotation

[α]_(D) 23 −20° (C 0.25, methanol)

Ultraviolet Absorption Spectrum

λ_(max) water 278 nm (ε 2224)

Solubility

Soluble water, methanol

Insoluble: n-hexane

Color Reaction

Positive: iodine vapor reaction, ceric sulfate reaction

Negative: Molish reaction, ferric chloride reaction

Thin Layer Chromatography (TLC)

Stationary phase Developing solvent Rf value Silica Gel 60 F₂₅₄n-butanol:acetic acid:water 0.50 (E. Merck) (4:1:2)

Infrared Spectrum

ν_(max) KBr: 3300, 2930, 2850, 1650, 1640, 1540, 1520, 1460, 1270, 1240,1050 (cm⁻¹)

¹H-NMR (500 MHz, CD₃OD, δ) 7.12 (1H, d, J=2 Hz), 6.86 (1H, dd, J=8 and 2Hz), 6.77 (1H, d, J=8 Hz), 5.35 (1H, dd, J=10 and 4 Hz), 5.07 (1H, d,J=4 Hz), 4.86 (1H, m), 4.51-4.45 (2H, m), 4.40-4.32 (3H, m), 4.31 (1H,d, J=2 Hz), 4.23 (1H, dd, J=11 and 4 Hz), 4.18-4.12 (2H, m), 3.81 (1H,d, J=l1 Hz), 3.73 (1H, dd, J=11 and 3 Hz), 3.67 (1H, br d, J=l1 Hz),3.37 (1H, t, J=10 Hz), 2.66 (1H,. m), 2.59 (1H, dd, J=16 and 4 Hz),2.56-2.47 (2H, m), 2.43 (1H, dd, J=16 and 9 Hz), 2.30-2.20 (4H, m),2.05-1.90 (3H, m), 1.75-1.61 (3H, m), 1.59 (2H, m), 1.37-1.23 (24H, m),1.06 (3H, d, J=7 Hz), 0.89 (3H, t, J=7 Hz)

¹³C-NMR (125 MHz, CD₃OD, δ): 176.7 (s), 176.2 (s), 174.6 (s), 174.0 (s),173.6 (s), 172.5 (s), 171.8 (s), 169.1 (s), 148.7 (s), 141.3 (s), 134.0(s), 127.0 (d), 124.0 (d), 118.3 (d), 75.9 (d), 72.0 (d), 71.3 (d), 70.7(d), 70.1 (d), 63.8 (t), 62.1 (d), 52.8 (t), 55.9 (d), 55.4 (d), 53.0(t), 52.1 (d), 39.3 (t), 39.1 (d), 38.4 (t), 36.8 (t), 34.8 (t), 33.4(t), 33.1 (t), 30.9 (t), 30.8 (t)×6, 30.7 (t), 30.6 (t), 30.5 (t), 30.4(t), 27.4 (t), 27.0 (t), 23.8 (t), 14.5 (q), 11.2 (q)

From the analysis of the above physical and chemical properties, and theresult of the further investigation of identification of chemicalstructure, the chemical structure of the WF 738D2 has been identifiedand assigned as follows.

EXAMPLE 4

1) Fermentation of Actinoplanes utahensis

A stock culture of Actinoplanes utahensis IFO-13244 is prepared andmaintained on agar slant. A loopful of the slant culture was inoculatedinto a seed medium consisting of starch 1%, sucrose 1%, glucose 1%,cotton seed flour 1%, peptone 0.5%, soy bean meal 0.5% and CaCO₃ 0.1%.The inoculated vegetative medium was incubated in a 225 ml-wide mouthErlenmeyer flask at 30° C. for about 72 hours on a rotary shaker.

This incubated vegetative medium was used directly to inoculate into aproduction medium (20 l) consisting of sucrose 2%, peanut powder 1%,K₂HPO₄ 0.12%, KH₂PO₄ 0.05% and MgSO₄.7H₂O 0.025%. The inoculatedproduction medium was allowed to ferment in a jar fermentor (30 l) at atemperature of 30° C. for about 80 hours. The fermentation medium wasstirred with conventional agitators at 250 rpm and aerated at 20 l/min.The vegetative mycelium was collected from the fermented broth byfiltration and once washed with water. The washed mycelium was directlyused to obtain the Deacyl WF 738A.

(2) Reaction Condition

To a solution of the WF 738A (420 mg) in water (105 ml) was added 1MNa-phosphate buffer (pH 5.8) (15 ml) and a washed mycelium ofActinoplanes utahensis IFO-13244 (10 g wet weight). The reaction wascarried out at 50° C. with stirring for 2 hours. The increase of theDeacyl WF 738A was monitored by HPLC indicated below.

From 420 mg of the WF 738A, 310 mg of the Deacyl WF 738A was formed inthe reaction mixture.

HPLC Condition

Column: YMC Pack ODS-AM AM303, S-5 120A (250 mm L.×4.6 mm I.D.; YMC Co.,Ltd.)

Eluent: 15% aqueous methanol—0.5% NaH₂PO₄.2H₂O

Flow rate: 1 ml/min

Detection: UV at 210 nm

Retention time: 11.6 minutes

(3) Isolation of the Deacyl WF 738A

The reaction mixture described above was filtrated with filter aid. Themycelial cake was discarded. The filtrate hus obtained was passedthrough a column (70 ml) of SEPABEADS SP-207 (Mitsubishi Chemical Co.,Ltd.) packed with water. The column was washed with water (210 ml) andthen eluted with 50% aqueous methanol (210 ml). The eluate wasconcentrated in vacuo to an aqueous solution (75 ml) and added 375 mg ofNaH₂PO₄.2H₂O. The solution was passed through a column (100 ml) of YMCGEL ODS-AM 120-S50 (YMC Co., Ltd.) packed with water. The column waseluted with 4% aqueous acetonitrile containing 0.5% NaH₂PO₄.2H₂O andelution was monitored by HPLC indicated before. The portioncorresponding to the Deacyl WF 738A was concentrated in vacuo to giveresidual water. This residue was further purified by preparative HPLCusing a YMC-packed column (ODS-AM SH-343 5AM S-5, YMC Co., Ltd., 250 mmL.×20 mm I.D.) with 15% aqueous methanol containing 0.5% NaH₂PO₄.2H₂O asa mobile phase and flow rate of 9.9 ml/min. The portion corresponding tothe Deacyl WF 738A was concentrated in vacuo to give residual water.This residue was passed through a column (50 ml) of DIAION HP-20(Mitsubishi Chemical Co., Ltd.) packed with water. The column was washedwith water (250 ml) and then eluted with 30% aqueous methanol. Theeluate was concentrated in vacuo and lyophilized to give 82 mg of theDeacyl WF 738A as a white powder.

The Deacyl WF 738A as obtained has the following physico-chemicalproperties.

HPLC Condition

Column: YMC Pack ODS-AM AM303, S-5 120A (250 mm L.×4.6 mm I.D.; YMC Co.,Ltd.)

Eluent: 15% methanol—0.5% NaH₂PO₄.2H₂O

Flow rate: 1 ml/min

Detection: UV at 210 nm

Retention time: 11.6 minutes

Appearance

white powder

Molecular Formula

C₃₄H₄₉N₈O₁₉SNa [free acid: C₃₄H₅₀N₈O₁₉S]

Molecular Weight

Molecular weight: 906 (free acid)

ESI-MS (negative): (m/z) 905 (M−H)⁻

ESI-MS (positive): (m/z) 907 (M+H)⁺

Melting point

163-168° C. (dec.)

Specific Rotation

[α]_(D) 23 −16° (C 0.5, water)

Ultraviolet Absorption Spectrum

λ_(max) water: 277 nm (ε1900)

Solubility

Soluble: water

Slightly soluble: methanol

Insoluble: ethyl acetate, n-hexane

Color Reaction

Positive: iodine vapor reaction, ceric sulfate reaction, ninhydrinreaction

Negative: Molish reaction, ferric chloride reaction

Thin Layer Chromatography (TLC)

Stationary phase Developing solvent Rf value Silica Gel 60 F₂₅₄ 70%aqueous isopropyl 0.67 (E. Merck) alcohol

Infrared Spectrum

ν_(max) KBR 3420, 2930, 1670, 1650, 1520, 1440, 1280, 1240, 1050 (cm⁻¹)

¹H-NMR (500MHz, D₂O, δ) 7.20 (1H, d, J=2 Hz), 6.99 (1H, dd, J=8 and 2Hz), 6.95 (1H , d, J=8 Hz), 5.38 (1H, d, J=3 Hz), 5.04 (1H, m), 4.92(1H, d J=6 Hz), 4.71 (1H, m), 4.63 (1H, m), 4.48 (1H, m), 4.42-4.22 (5H,m), 4.12 (1H, m), 4.07-4.01 (2H, m), 3.93-3.87 (3H, m), 3.41 (1H, m),2.78-2.68 (2H, m), 2.60-2.50 (2H, m), 2.45-2.34 (3H, m), 2.12 (1H, m),2.02 (1H, m), 1.02 (3H, d, J=6 Hz)

¹³C-NMR (125 MHz, D₂O, δ): 178.4 (s), 176.7 (s), 174.6 (s), 174.4 (s),173.8 (s), 171.7 (s), 171.4 (s), 149.4 (s), 141.5 (s), 132.5 (s), 130.7(d), 126.6 (d), 120.2 (d), 78.3 (d), 76.8 (d), 74.5 (d), 73.0 (d), 72.9(d), 71.7 (d), 69.5 (d), 64.0 (t), 63.9 (d), 59.7 (d), 58.3 (t), 57.5(d), 57.0 (d), 55.4 (d), 54.9 (t), 41.8 (t), 41.6 (t), 40.0 (t), 39.9(d), 34.1 (t), 13.4 (q)

From the analysis of the above physical and chemical properties, and theresult of the further investigation of identification of chemicalstructure, the chemical structure of the Deacyl WF 738A has beenidentified and assigned as follows.

EXAMPLE 5

(1) Fermentation of Actinoplanes Utahensis

The fermentation of Actinoplanes utahensis was carried out according toa similar manner to that of Example 4.

(2) Reaction Condition

To a solution of the WF 738B (5 g) in water (2.1 l) was added 1MNa-phosphate buffer (pH 5.8) (300 ml) and a washed mycelium ofActinoplanes utahensis IFO-13244 (300 g wet weight). The reaction wascarried out at 50° C. with stirring for 1.5 hours. The increase of theDeacyl WF 738B was monitored by HPLC indicated below.

From 5 g of the WF 738B, 3.9 g of the Deacyl WF 738B was formed in thereaction mixture.

HPLC Condition

Column: YMC Pack ODS-AM AM303, S-5 120A (250 mm L.×4.6 mm I.D.; YMC Co.,Ltd.)

Eluent: 15% aqueous methanol—0.5% NaH₂PO₄.2H₂O

Flow rate: 1 ml/min

Detection: UV at 210 nm

Retention time: 7.2 minutes

(3) Isolation of the Deacyl WF 738B

The reaction mixture described above was filtrated with a filter aid.The mycelial cake was discarded. The filtrate thus obtained was passedthrough a column (800 ml) of SEPABEADS SP-207 (Mitsubishi Chemical Co.,Ltd.) packed with water. The column was washed with water (2.4 l) andthen eluted with 50% aqueous methanol (2 l). The eluate was concentratedin vacuo to an aqueous solution (620 ml) and added 3.1 g ofNaH₂PO₄.2H₂O. The solution was passed through a column (2 l) of YMC GELODS-AM 120-S50 (YMC Co., Ltd) packed with water. The column was elutedwith 2% aqueous acetonitrile containing 0.5% NaH₂PO₄.2H₂O and elutionwas monitored by HPLC indicated before. The portion corresponding to theDeacyl WF 738B was concentrated in vacuo to give residual water. Thisresidue was passed through a column (2 l) of YMC GEL ODS-AM 120-S50 (YMCCo., Ltd.) packed with water. The column was washed with water (10 l)and then eluted with 20% aqueous methanol. The eluate was concentratedin vacuo and lyophilized to give 1.15 g of the Deacyl WF 738B as a whitepowder.

The Deacyl WF 738B as obtained has the following physico-chemicalproperties.

HPLC Condition

Column: YMC Pack ODS-AM AM303, S-5 120A (250 mm L.×4.6 mm I.D.; YMC Co.,Ltd.)

Eluent: 15% methanol—0.5% NaH₂PO₄.2H₂O

Flow rate: 1 ml/min

Detection: UV at 210 nm

Retention time: 7.7 minutes

Appearance

white powder

Molecular Formula

C₃₃H₄₇N₈O₁₉SNa [free acid: C₃₃H₄₈N₈O₁₉S]

Molecular Weight

Molecular Weight: 892 (free acid)

ESI-MS (negative): (m/z) 891 (M−H)⁻

ESI-MS (positive): (m/z) 893 (M+H)⁺

Melting Point

154-159° C. (dec.)

Specific Rotation

[α]_(D) 23 −19° (C 0.75, water)

Ultraviolet Absorption Spectrum

λ_(max) water: 277 nm (ε 2187)

Solubility:

Soluble: water

Slightly soluble: methanol

Insoluble: ethyl acetate, n-hexane

Color Reaction:

Positive: iodine vapor reaction, ceric sulfate reaction, ninhydrinreaction

Negative: Molish reaction, ferric chloride reaction

Thin Layer Chromatography (TLC)

Stationary phase Developing solvent Rf value Silica Gel 60 F₂₅₄ 70%aqueous isopropyl 0.33 (E. Merck) alcohol

Infrared Spectrum λ_(max) KBr: 3440, 2940, 1670, 1630, 1520, 1450, 1270,1050 (cm⁻¹)

¹H-NMR (500 MHz, D₂O, δ) 7.20 (1H, d, J=2 Hz), 6.97 (1H, dd, J=8 and 2Hz), 6.95 (1H, d, J=8 Hz), 5.38 (1H, d, J=3.5 Hz), 5.06 (1H, m), 4.93(1H, d J=6 Hz), 4.71 (1H, m), 4.62 (1H, m), 4.51-4.47 (2H, m), 4.43 (1H,d, J=2 Hz), 4.33 (1H, m), 4.27-4.22 (2H, m), 4.12 (1H, m), 4.08-4.02(2H, m), 3.91-3.87 (2H, m), 3.82-3.78 (2H, m), 2.78-2.68 (2H, m), 2.54(1H, m), 2.43-2.30 (4H, m), 2.16-1.97 (3H, m)

¹³C-NMR (125 MHz, D₂O, δ) 178.4 (s), 176.8 (s), 174.6 (s), 174.4 (s),173.8 (s), 171.7 (s), 171.2 (s), 149.5 (s), 141.5 (s), 132.5 (s), 130.7(d), 126.5 (d), 120.2 (d), 78.2 (d), 75.2 (d), 74.6 (d), 72.9 (d), 72.8(d), 71.8 (d), 70.2 (d), 64.0 (t), 63.9 (d), 59.7 (d), 58.4 (t), 57.5(d), 57.0 (d), 55.4 (d), 48.6 (t), 41.7 (t), 41.6 (t), 39.9 (t), 35.4(t), 34.1 (t)

From the analysis of the above physical and chemical properties, and theresult of the further investigation of identification of chemicalstructure, the chemical structure of the Deacyl WF 738B has beenidentified and assigned as follows.

The Starting Compound in the following Example 6 to 17 and The ObjectCompounds (6) to (17) in the following Example 6 to 17 are illustratedby chemical formulae as below.

The Starting Compound (the same in Example 6 to 17)

The Object Compounds (6) to (17)

The Object Compound (X) [e.g. The Object Compound (6)] means the objectcompound of Example (X) [e.g. Example 6].

The Object Compound R₁ 6

7

8

9

10

11

12

13

14

15

16

17

EXAMPLE 6

To a solution of The Starting Compound (0.1 g) and1-[4-[4-(4-hexyloxyphenyl)piperazin-1-yl]benzoyloxy]benzotriazole (0.062g) in dimethylformamide (2 ml) was added N,N-diisopropylethylamine(0.029 ml), and stirred for 5 hours at ambient temperature. The reactionmixture was pulverized with ethyl acetate. The precipitate was collectedby filtration, and dried under reduced pressure. The powder was added topH 6.86 phosphate buffer, and purified by preparative HPLC utilizing ODSresin which was eluted with a solvent system comprised ofacetonitrile-pH 6.86 phosphate buffer (38:62) at a flow rate of 80ml/minutes using a Shimadzu LC-8A pump. The column was monitored by a UVdetector set at 210 nm. The fractions containing The Object Compoundwere combined, and evaporated under reduced pressure to removeacetonitrile. The residue was adjusted to pH 6.5 with saturated sodiumbicarbonate aqueous solution, and subjected to column chromatography onODS (YMC-gel ODS-AM.S-50) (Trademark: prepared by Yamamura ChemicalLab.), and washed with water, and eluted with 60% acetonitrile aqueoussolution. The fractions containing The Object Compounds were combined,and evaporated under reduced pressure to remove acetonitrile. Theresidue was lyophilized to give The Object Compound (6) (105 mg).

IR (KBr): 3361.3, 1668.1, 1627.6, 1510.0, 1236.1, 1045.2 cm⁻¹

NMR (DMSO-d₆, δ): 0.88 (3H, t, J=6.6 Hz), 1.2-1.5 (6H, m), 1.6-2.6 (12H,m), 3.0-5.6 (35H, m), 6.6-7.15 (11H, m), 7.2-7.4 (2H, m), 7.56 (1H, d,J=9.7 Hz), 7.84 (2H, d, J=8.6 Hz), 8.19 (1H, d, J=9.2 Hz), 8.45 (1H, d,J=7.2 Hz), 8.69 (1H, d, J=7.2 Hz)

MASS (m/z): 1256 (M−Na)⁺

Elemental Analysis Calcd. for C₅₆H₇₅N₁₀NaO₂₁S.7H₂O: C, 47.86; H, 6.38;N, 9.97.

Found: C, 47.81; H, 6.56; N, 10.46.

The following compounds [Example 7 to 17] were obtained according to asimilar manner to that of Example 6.

EXAMPLE 7

The Object Compound (7)

IR (KBr): 3361, 1621.8, 1538.9, 1508.1, 1257.4, 1047.2 cm⁻¹

NMR (DMSO-d₆, δ): 0.91 (3H, t, J=7.0 Hz), 1.25-1.55 (4H, m), 1.65-2.60(12H, m), 3.50-4.50 (15H, m), 4.06 (2H, d, J=6.5 Hz), 4.75 (8H, m), 5.42(1H, m), 5.54 (1H, d, J=5.9 Hz), 6.70 (1H, d, J=8.2 Hz), 6.76 (1H, dd,J=8.2 and 1.5 Hz), 6.79 (1H, s), 6.96 (1H, d, J=1.5 Hz), 7.12 (2H, d,J=8.8 Hz), 7.23 (1H, s), 7.33 (1H, d, J=8.2 Hz), 7.45-7.65 (1H, m), 7.55(1H, s), 7.85 (2H, d, J=8.8 Hz), 8.00 (2H, d, J=8.7 Hz), 8.06 (2H, d,J=8.7 Hz), 8.19 (1H, d, J=8.7 Hz), 8.69 (1H, d, J=7.0 Hz), 8.73 (1H, s),8.87 (1H, d, J=7.3 Hz)

MASS (m/z): 1224.4 (M−Na)⁺

Elemental Analysis Calcd. for C₅₄H₆₆N₉NaO₂₂S.7H₂O: C, 47.19; H, 5.87; N,9.17.

Found C, 47.42; H, 5.62; N, 9.09.

EXAMPLE 8

The Object Compound (8)

IR (KBr): 3345.9, 1664.3, 1629.6, 1519.6, 1247.7, 1047.2 cm⁻¹

NMR (DMSO-d₆, δ): 0.91 (3H, t, J=7.0 Hz), 1.25-1.55 (4H, m), 1.65-2.65(12H, m), 3.45-5.50 (27H, m), 6.70 (1H, d, J=8.2 Hz), 6.77 (1H, d, J=8.2Hz), 6.86 (1H, s), 6.90-7.15 (2H, m), 7.04 (2H, d, J=8.7 Hz), 7.22 (1H,s), 7.50-7.95 (7H, m), 7.67 (2H, d, J=8.7 Hz), 8.01 (2H, d, J=8.5 Hz),8.15-8.3 (1H, m), 8.5-9.0 (3H, m)

MASS (m/z): 1234 (M−Na)⁺

Elemental Analysis Calcd. for C₅₇H₆₉N₈NaO₂₁S.8H₂O: C, 48.85; H, 6.11; N,8.00.

Found: C, 48.78; H, 5.84; N, 7.92.

EXAMPLE 9

The Object Compound (9)

IR (KBr): 3340.1, 1629.6, 1515.8, 1444.4, 1257.4, 1047.2 cm⁻¹

NMR (DMSO-d₆, δ): 0.89 (3H, t, J=6.9 Hz), 1.2-1.55 (6H, m), 1.65-2.60(12H, m), 3.50-4.55 (15H, m), 4.08 (2H, d, J=6.5 Hz), 4.7-5.3 (8H, m),5.43 (1H, m), 5.54 (1H, d, J=5.9 Hz), 6.70 (1H, d, J=8.1 Hz), 6.76 (1H,d, J=8.1 Hz), 6.85 (1H, s), 6.96 (1H, s), 7.13 (2H, d, J=8.9 Hz), 7.21(1H, s), 7.33 (1H, d, J=8.3 Hz), 7.55 (1H, d, J=8.9 Hz), 7.97 (2H, d,J=8.9 Hz), 8.07 (1H, d, J=9.5 Hz), 8.12 (1H, d, J=9.5 Hz), 8.20 (1H, d,J=9.0 Hz), 8.6-8.8 (1H, m), 8.73 (1H, s), 8.93 (1H, d, J=6.6 Hz)

MASS (m/z): 1256 (M−Na)⁺

Elemental Analysis Calcd. for C₅₄H₆₇N₁₀NaO₂₁S₂.6H₂O: C, 46.75; H, 5.73;N, 10.10.

Found: C, 46.57; H, 5.66; N, 10.03.

EXAMPLE 10

The Object Compound (10)

IR (KBr): 3340, 1666.2, 1629.6, 1508.1, 1255.4, 1045.2 cm⁻¹

NMR (DMSO-d₆, δ): 0.86 (3H, t, J=6.6 Hz), 1.2-1.55 (10H, m), 1.6-2.60(12H, m), 3.50-4.55 (15H, m), 4.02 (2H, d, J=6.4 Hz), 4.7-5.3 (8H, m),5.40 (1H, m), 5.48 (1H, d, J=5.9 Hz), 6.70 (1H, d, J=8.1 Hz), 6.76 (1H,d, J=8.1 Hz), 6.81 (1H, s), 6.95 (1H, s), 6.97 (2H, d, J=8.8 Hz), 7.28(1H, s), 7.33 (1H, d, J=8.3 Hz), 7.55 (1H, d, J=8.4 Hz), 7.86 (2H, d,J=8.8 Hz), 8.18 (1H, d, J=9.3 Hz), 8.56 (1H, d, J=8.1 Hz), 8.6-8.8 (1H,m), 8.72 (1H, s)

MASS (m/z): 1124 (M−Na)⁺

EXAMPLE 11

The Object Compound (11)

IR (KBr): 3340, 1666.2, 1629.6, 1519.6, 1047.2 cm⁻¹

NMR (DMSO-d₆, δ): 0.87 (3H, t, J=7.0 Hz), 1.2-1.5 (4H, m), 1.6-2.6 (12H,m), 3.50-4.55 (15H, m), 4.08 (2H, d, J=6.5 Hz), 4.7-5.3 (8H, m), 5.42(1H, m), 5.53 (1H, d, J=5.9 Hz), 6.71 (1H, d, J=8.1 Hz), 6.76 (1H, d,J=8.1 Hz), 6.85 (1H, s), 6.96 (1H, s), 7.14 (2H, d, J=8.9 Hz), 7.27 (1H,s), 7.32 (1H, d, J=8.0 Hz), 7.56 (1H, d, J=8.7 Hz), 7.90 (2H, d, J=8.9Hz), 7.98 (4H, s), 8.20 (1H, d, J=9.5 Hz), 8.6-8.8 (2H, m), 8.73 (1H,s), 8.86 (1H, s)

MASS (m/z): 1281 (M−Na)⁺

Elemental Analysis Calcd. for C₅₅H₆₆N₁₁NaO₂₁S₂.7H₂O: C, 46.18; H, 5.64;N, 10.77.

Found: C, 46.09; H, 5.72; N, 10.60.

EXAMPLE 12

The Object Compound (12)

IR (KBr): 3340, 1666.2, 1650.8, 1631.5, 1535.1, 1511.9, 1442.5, 1247.7cm⁻¹

NMR (DMSO-d₆, δ): 1.00 (3H, t, J=7.4 Hz), 1.6-2.60 (12H, m), 3.50-4.55(15H, m), 4.00 (2H, d, J=6.5 Hz), 4.7-5.3 (8H, m), 5.44 (1H, m), 5.55(1H, d, J=5.9 Hz), 6.71 (1H, d, J=8.1 Hz), 6.76 (1H, d, J=8.1 Hz), 6.86(1H, s), 6.96 (1H, s), 7.07 (2H, d, J=8.8 Hz), 7.22 (1H, s), 7.34 (1H,d, J=8.4 Hz), 7.56 (1H, d, J=8.6 Hz), 7.73 (2H, d, J=8.8 Hz), 7.86 (2H,d, J=8.4 Hz), 8.18 (1H, d, J=9.3 Hz), 8.05-8.3 (7H, m), 8.6-8.8 (1H, m),8.73 (1H, s), 8.94 (1H, d, J=7.0 Hz)

MASS (m/z): 1289 (M−Na)³⁰

Elemental Analysis Calcd. for C₅₇H₆₅N₁₀NaO₂₁S₂.7H₂O: C, 47.56; H, 5.53;N, 9.73.

Found: C, 47.86; H, 5.60; N, 9.70.

Example 13

The Object Compound (13)

IR (KBr): 3390.2, 2954.4, 2933.2, 1629.6, 1519.6, 1440.6, 1255.4, 1049.1cm⁻¹

NMR (DMSO-d₆, δ) 1.80-1.00 (3H, m), 1.25-1.60 (4H, m), 1.60-1.70 (12H,m), 3.50-4.60 (17H, m), 4.78 (2H, d, J=6.1 Hz), 4.95 (2H, d, J=6.3 Hz),5.05-5.35 (4H, m), 5.42 (1H, s), 5.54 (2H, d, J=5.8 Hz), 6.71 (2H, d,J=8.2 Hz), 6.77(2H, d, J=8.2 Hz), 6.85 (1H, s), 6.96 (1H, s), 7.08 (2H,d, J=8.8 Hz), 7.24 (1H, s), 7.35 (2H, d, J=8.2 Hz), 7.58 (2H, d, J=8.4Hz), 7.81 (2H, d, J=8.4 Hz), 7.92 (2H, d, J=8.8 Hz), 7.99 (2H, d, J=8.4Hz), 8.21 (1H, d, J=8.7 Hz), 8.40 (1H, s), 8.69 (1H, d, J=7.0 Hz), 8.73(1H, s), 8.83 (1H, d, J=7.3 Hz)

MASS (m/z): 1240.30 (M−Na)³⁰

Elemental Analysis Calcd. for C₅₄H₆₆N₉NaO₂₁S₂.6H₂O: C, 47.26; H, 5.73;N, 9.19.

Found: C, 47.34; H, 5.58; N, 9.20.

EXAMPLE 14

The Object Compound (14)

IR (KBr): 3365, 1660.4, 1623.8, 1517.7, 1247.7, 1047.2 cm⁻¹

NMR (DMSO-d₆, δ): 0.91 (3H, t, J=7.1 Hz), 1.2-1.55 (4H, m), 1.6-2.60(12H, m), 3.50-4.55 (15H, m), 4.01 (2H, d, J=6.4 Hz), 4.7-5.3 (8H, m),5.39 (1H, m), 5.53 (1H, d, J=5.9 Hz), 6.70 (1H, d, J=8.1 Hz), 6.75 (1H,d, J=14 Hz), 6.76 (1H, d, J=8.1 Hz), 6.85 (1H, s), 6.95 (1H, s), 7.02(2H, d, J=8.8 Hz), 7.30 (1H, s), 7.33 (1H, d, J=8.3 Hz), 7.40 (1H, d,J=14 Hz), 7.5-7.8 (7H, m), 8.23 (1H, d, J=8.7 Hz), 8.52 (1H, d, J=7.8Hz), 8.6-8.8 (1H, m), 8.71 (1H, s)

MASS (m/z) 1184 (M−Na)³⁰

EXAMPLE 15

The Object Compound (15)

IR (KBr): 3390.2, 2954.4, 2933.2, 1629.6, 1519.6, 1440.6, 1255.4, 1049.1cm⁻¹

NMR (DMSO-d₆, δ) 1.80-1.00 (3H, m), 1.20-1.55 (6H, m), 1.60-2.60 (12H,m), 3.50-4.60 (17H, m), 4.78 (2H, d, J=6.1 Hz), 4.95 (2H, d, J=6.3 Hz),5.05-5.35 (4H, m), 5.42 (1H, s), 5.54 (2H, d, J=6.0 Hz), 6.71 (2H, d,J=8.3 Hz), 6.77 (2H, d, J=8.3 Hz), 6.85 (1H, s), 6.96 (1H, s), 7.08 (2H,d, J=8.8 Hz), 7.24 (1H, s), 7.33 (2H, d, J=8.3 Hz), 7.56 (2H, d, J=9.9Hz), 7.81 (2H, d, J=8.4 Hz), 7.91 (2H, d, J=8.8 Hz), 7.99 (2H, d, J=8.4Hz), 8.21 (1H, d, J=8.3 Hz), 8.40 (1H, s), 8.69 (1H, d, J=7.0 Hz), 8.73(1H, s), 8.81 (1H, d, J=7.1 Hz)

MASS (m/z): 1253.55 (M−Na)³⁰

Elemental Analysis Calcd. for C₅₅H₆₈N₉NaO₂₁S₂.6H₂O: C, 47.65; H, 5.82;N, 9.09.

Found: C, 47.67; H, 5.65; N, 9.06.

EXAMPLE 16

The Object Compound (16)

IR (KBr): 3425.0, 2933.2, 2861.8, 1633.4, 1533.1, 1444.4, 1251.6 cm⁻¹

NMR (DMSO-d₆δ): 1.80-1.00 (3H, m), 1.20-1.55 (6H, m), 1.60-2.60 (12H,m), 3.50-4.60 (17H, m), 4.78 (2H, d, J=6.0 Hz), 4.95 (2H, d, J=6.3 Hz),5.05-5.35 (4H, m), 5.43 (1H, s), 5.55 (2H, d, J=5.9 Hz), 6.71 (2H, d,J=8.2 Hz), 6.77 (2H, d, J=8.2 Hz), 6.85 (1H, s), 6.96 (1H, s), 7.04 (2H,d, J=8.8 Hz), 7.23 (1H, s), 7.33 (2H, d, J=7.1 Hz), 7.56 (2H, d, J=8.2Hz), 7.67 (2H, d, J=8.8 Hz), 8.04 (4H, s), 8.21 (1H, d, J=8.3 Hz), 8.27(1H, s), 8.69 (1H, d, J=7.0 Hz), 8.73 (1H, s), 8.89 (1H, d, J=7.1 Hz)

MASS (m/z): 1254.07 (M−Na)³⁰

Elemental Analysis Calcd. for C₅₅H₆₈N₉NaO₂₁S₂.6H₂O: C, 47.65; H, 5.82;N, 9.09.

Found: C, 47.46; H, 5.71; N, 9.07.

EXAMPLE 17

The Object Compound (17)

IR (KBr): 3467.4, 2964.1, 2939.0, 1629.6, 1517.7, 1440.6, 1247.7, 1047.2cm⁻¹

NMR (DMSO-d₆, δ): 1.00 (3H, t, J=7.4 Hz), 1.60-2.70 (12H, m), 3.45-4.50(17H, m), 4.50-5.40 (10H, m), 6.65-6.95 (3H, m), 6.96 (1H, s), 7.06 (2H,d, J=8.8 Hz), 7.23 (2H, br s), 7.56 (1H, m), 7.71 (2H, d, J=8.8 Hz),7.75-7.95 (4H, m), 7.95-8.15 (4H, m), 8.20 (1H, m), 8.35-9.0 (4H, m)

MASS (m/z): 1288.14 (M−Na)³⁰

Elemental Analysis Calcd. for C₅₈H₆₆N₉NaO₂₁S₂.7H₂O: C, 48.43; H, 5.61;N, 8.76.

Found: C, 48.69; H, 5.41; N, 8.67.

EXAMPLE 18

(1) Fermentation of Streptomyces sp. No. 6907

A stock culture of Streptomyces sp. No. 6907 is prepared and maintainedon agar slant. A loopful of the slant culture was inoculated into 60 mlof sterilized seed medium consisting of maltose 3%, dried yeast 1%,CaCO₃ 0.5% in a 225-ml Erlenmeyer flask. The flask was incubated at 30°C. for 3 days on a rotary shaker (220 rpm, 5.1 cm-throw) and theninoculated (0.1%) into 160 ml of sterilized seed medium consisting ofmaltose 3%, dried yeast 1%, CaCO₃ 0.5%, Adekanol LG-109 (Asahi DenkaCo., Ltd.) 0.1% and Silicone KM-70 (Shin-Etsu chemical Co., Ltd.) 0.1%in each of seven 500-ml Erlenmeyer flasks. And the flasks were incubatedat 30° C. for 2 days on a rotary shaker (220 rpm, 5.1 cm-throw).

The resultant seed culture was then inoculated (5%) into 20 liters ofsterilized production medium consisting of maltose 8%, soybean meal 2%,wheat germ 2%, potato protein 2%, CaCO₃ 0.5%, Adekanol LG-109 0.1% andSilicone KM-70 0.1% in a 30-liter-jar fermenter. The fermentation wascarried out at 30° C. for 5 days under aeration of 20 liters/minute andagitation of 200 rpm. The fermentation broth was directly used to obtainthe Deacyl WF 738C.

(2) Fermentation of Coleophoma Crateriformis No. 738

The formentaion of Example 18 was carried out according to a similarmanner to that of Example 1.

(3) Preparation of the Crude WF 738 C

After the above culture was completed, an equal volume of acetone wasadded to the cultured broth (120 liters). The mixture was allowed tostand for about an hour with stirring at room temperature. The resultantmixture was filtrated with an aid of diatomaceous earth. The filtratewas diluted with an equal volume of water and passed through a column(12 liters) of DIAION HP-20 (Mitsubishi Chemical Co., Ltd.) packed withwater. The column was washed with water (40 liters) and 50% aqueousmethanol (40 liters) and then eluted with 90% aqueous methanol (24liters). The eluate was concentrated in vacuo to an aqueous solution(1300 ml).

(4) Reaction Condition

300 ml of 1M Na-phosphate buffer (pH5.8) and 700 ml of the fermentationbroth of Streptomyces sp. No. 6907 were added to the solution obtainedabove. This reaction mixture was adjusted to 6 liters with water. Thereaction was carried out at 50° C. with stirring for an hour. Theincrease of the Deacyl WF 738C was monitored by analytical HPLCindicated below.

Analytical HPLC Condition

Column: TSKgel Amide-80 (250×4.6 mm I.D., TOSOH Co., Ltd.)

Eluent: 70% aqueous acetonitrile containing 0.05% Trifluoroacetic acid

Flow rate: 1 ml/min.

Detection: UV at 210 nm

Retention time: 9.2 minutes

(5) Isolation of the Deacyl WF 738C

The reaction mixture described above was filtrated with an aid ofdiatomaceous earth. The mycelial cake was discarded. The filtrate thusobtained was passed through a column (3.2 liters) of SEPABEADS SP-207(Mitsubishi Chemical Co., Ltd.) packed with water. The column was washedwith water (9.5 liters) and then eluted with 50% aqueous methanol (7liters). The eluate was concentrated in vacuo to an aqueous solution(1300 ml). This solution was passed through a column (8 liters) of YMCGEL (ODS-AM 120-S50, YMC Co., Ltd.) packed with water. The column waswashed with 4% aqueous acetonitrile containing 0.5% NaH₂PO₄.2H₂O (63liters) and eluted with 8% aqueous acetonitrile containing 0.5%NaH₂PO₄.2H₂O. Elution was monitored by analytical HPLC indicated before.Fractions containing the Deacyl WF 738C was concentrated in vacuo togive residual water. This residue was passed through a column (300 ml)of YMC GEL (ODS-AM 120-S50) packed with water. The column was washedwith water (1500 ml) and then eluted with 20% aqueous methanol. Theeluate was concentrated in vacuo and lyophilized to give 2 g of theDeacyl WF 738C as a pale yellowish powder. This powder was dissolved ina small volume of water and passed through a column (2 liters) of YMCGEL (ODS-AM 120-S50) packed with water. The column was eluted with 10%aqueous methanol. Fractions containing the Deacyl WF 738C wasconcentrated in vacuo and further purified by preparative HPLC, usingTSKgel Amide-80 column (300×21.5 mm I.D., TOSOH Co., Ltd.) with 75%aqueous acetonitrile containing 10 mM NaH₂PO₄.2H₂O as a mobile phase anda flow rate of 5.6 ml/minute. Fractions containing the Deacyl WF 738Cwere collected and concentrated in vacuo to give residual water. Thisresidue was passed through YMC-packed column (ODS-AM SH-343-5AM S-5,250×20 mm I.D., YMC Co., Ltd.) equilibrated with water. The column waswashed with water (400 ml) and then eluted with 20% aqueous methanol ata flow rate of 9.9 ml/minute. The eluate was concentrated in vacuo andlyophilized to give 42.2 mg of the Deacyl WF 738C as a white powder.

The Deacyl WF 738C as obtained has the following physico-chemicalproperties.

HPLC Condition

Column: YMC Pack ODS-AM AM303, S-5 120A (250 mm L.×4.6 mm I.D.; YMC Co.,Ltd.)

Eluent: 15% methanol—0.5% NaH₂PO₄.2H₂O

Flow rate: 1 ml/min

Detection: WV at 210 nm

Retention time: 10.2 minutes

Appearance

white powder

Molecular Formula

C₃₄H₅₀N₈O₁₈S

Molecular Weight

Molecular weight: 890.88

ESI-MS (negative): (m/z) 889 (M−H)⁻

ESI-MS (positive): (mlz) 891 (M+H)⁺

Melting Point

215-223° C. (dec.)

Specific Rotation

[α]_(D) 23−16°(C 1.0, water)

Ultraviolet Absorption Spectrum

λ_(max) water: 276 nm (ε 1200)

Solubility

Soluble: water

Slightly soluble: methanol

Insoluble: ethyl acetate, n-hexane

Color Reaction

Positive: iodine vapor reaction, ceric sulfate reaction

Negative: Molish reaction, ferric chloride reaction

Thin Layer Chromatography (TLC)

Stationary phase Developing solvent Rf value Silica Gel 60 F₂₅₄ 70%aqueous isopropyl 0.67 (E. Merck) alcohol

Infrared Spectrum

ν_(max) KBr: 3370, 2950, 1670, 1630, 1520, 1450, 1270, 1240, 1050 (cm⁻¹)

¹H-NMR (500 MHz, D₂O, δ): 7.20 (1H, d, J=2 Hz), 6.98 (1H, dd, J=8 and 2Hz), 6.96 (1H, d, J=8 Hz), 5.56 (1H, m), 5.07 (1H, m), 4.99 (1H, d, J=6Hz), 4.71 (1H, m), 4.62 (1H, m), 4.50-4.43 (2H, m), 4.35 (1H, m),4.33-4.26 (3H, m), 4.07(1H, m), 3.97 (1H, m), 3.93-3.82 (3H, m), 3.47(1H, m), 2.75-2.65 (2H, m), 2.62-2.47 (3H, m), 2.36 (1H, m), 2.24-2.10(2H, m), 2.04-1.90 (3H, m), 1.04 (3H, d, J=7 Hz)

¹³C-NMR (125 MHz, D₂O, δ): 178.5 (s), 176.7 (s), 174.7 (s), 174.7 (s),173.3 (s), 172.0 (s), 170.9 (s), 149.4 (s), 141.5 (s), 132.5 (s), 130.7(d), 126.5 (d), 120.2 (d), 77.3 (d), 75.0 (d), 73.5 (d), 73.0 (d), 71.8(d), 70.0 (d), 64.5 (t), 63.9 (d), 59.5 (d), 58.3 (t), 57.5 (d), 56.9(d), 54.9 (t), 54.3 (d), 41.6 (t), 41.5 (t), 40.0 (d), 39.9 (t), 31.6(t), 29.1 (t), 13.3 (q)

From the analysis of the above physical and chemical properties, and theresult of the further investigation of identification of chemicalstructure, the chemical structure of the Deacyl WF 738C has beenidentified and assigned as follows.

EXAMPLE 19

(1) Fermentation of Streptomyces sp. No. 6907

The fermentation of Streptomyces sp. No. 6907 of Example 19 was carriedout according to a similar manner to that of Example 18.

(2) Fermentation of Coleophoma Crateriformis No. 738

The fermentation of Example 19 was carried out according to a similarmanner to that of Example 1.

(3) Preparation of the Crude Deacyl WF 738F

After the above culture was completed, an equal volume of acetone wasadded to the cultured broth (180 liters). The mixture was allowed tostand for about an hour with stirring at room temperature. The resultantmixture was filtrated with an aid of diatomaceous earth. The filtratewas diluted with an equal volume of water and passed through a column(18 liters) of DIAION HP-20 (Mitsubishi Chemical Co., Ltd.) packed withwater. The column was washed with water (60 liters) and 50% aqueousmethanol (60 liters) and then eluted with 90% aqueous methanol (38liters). The eluate was concentrated in vacuo to an aqueous solution (2liters).

450 ml of 1M Na-phosphate buffer (pH5.8) and 1 liter of the fermentationbroth of Streptomyces sp. No. 6907 were added to this solution (2liters). This reaction mixture was adjusted to 9 liters with water. Thereaction was carried out at 50° C. with stirring for an hour.

(4) Isolation of the Deacyl WF 738F

The reaction mixture described above was filtrated with an aid ofdiatomaceous earth. The mycelial cake was discarded. The filtrate thusobtained was passed through a column (5 liters) of SEPABEADS SP-207(Mitsubishi Chemical Co., Ltd.) packed with water. The column was washedwith water (15 liters) and then eluted with 50% aqueous methanol (12liters). The eluate was concentrated in vacuo to an aqueous solution (2liters). This solution was passed through a column (12 liters) of YMCGEL (ODS-AM 120-S50, YMC Co., Ltd.) packed with water. The column waseluted with 4% aqueous acetonitrile containing 0.5% NaH₂PO₄.2H₂O.Elution was monitored by analytical HPLC indicated below. Fractionscontaining the Deacyl WF 738F was concentrated in vacuo to give residualwater. This residue was passed through a column (4 liters) of YMC GEL(ODS-AM 120-S50) packed with water. The column was eluted with 6.5%aqueous methanol. Fractions containing the Deacyl WF 738F wasconcentrated in vacuo and passed through a column (350 ml) of YMC GEL(ODS-AM 120-S50) packed with water. The column was eluted with 7%aqueous methanol. Fractions containing the Deacyl WF 738F wasconcentrated in vacuo and lyophilized to give a white powder (96.6 mg).This powder was dissolved in 400 ml of water and allowed to stand atroom temperature to give 28.9 mg of the Deacyl WF 738F substance ascolorless prisms.

Analytical HPLC Condition

Column: TSKgel Amide-80 (250×4.6 mm I.D., TOSOH Co., Ltd.)

Eluent: 70% aqueous acetonitrile containing 0.05% Trifluoroacetic acid

Flow rate: 1 ml/min.

Detection: UV at 210 nm

Retention time: 10.6 minutes

The Deacyl WF 738F as obtained has the following physico-chemicalproperties.

HPLC Condition

Column: YMC Pack ODS-AM AM303, S-5 120A (250 mm L.×4.6 mm I.D.; YMC Co.,Ltd.)

Eluent: 15% methanol—0.5% NaH₂PO₄2H₂O

Flow rate: 1 ml/min

Detection: UV at 210 nm

Retention time: 6.9 minutes

Appearance

Colorless prisms

Molecular Formula

C₃₃H₄₈N₈O₁₈S

Molecular Weight

Molecular weight: 876.85

ESI-MS (negative): (m/z) 875 (M−H)⁻

ESI-MS (positive): (m/z) 877 (M+H)⁺

Melting Point

200-212° C. (dec.)

Specific Rotation

[α]_(D) 23−19° (C 0.7, water)

Ultraviolet Absorption Spectrum

λ_(max) water: 277.5 nm (ε 1800)

Solubility

Soluble: water

Slightly soluble: methanol

Insoluble: ethyl acetate, n-hexane

Color Reaction

Positive: iodine vapor reaction, ceric sulfate reaction

Negative: Molish reaction, ferric chloride reaction

Thin Layer Chromatography (TLC)

Stationary phase Developing solvent Rf value Silica Gel 60 F₂₅₄ 70%aqueous isopropyl 0.64 (E. Merck) alcohol

Infrared Spectrum

ν_(max) KBr: 3360, 2950, 1670, 1630, 1540, 1515, 1450, 1270, 1240, 1040(cm⁻¹)

¹H-NMR (500 MHz, D₂O, δ): 7.20 (1H, d, J=2 Hz), 6.99 (1H, dd, J=8 and 2Hz), 6.96 (1H, d, J=8 Hz), 5.56 (1H, m), 507 (1H, m), 5.00 (1H, d, J=6Hz), 4.72 (1H, m), 4.62 (1H, m), 4.49-4.43 (3H, m), 4.31-4.27 (2H, m),4.16 (1H, d, J=5 Hz), 4.07 (1H, m), 3.94-3.80 (5H, m), 2.75-2.65 (2H,m), 2.54 (1H, m), 2.48 (1H, m), 2.40-2.32 (2H, m), 2.20 (1H, m), 2.14(1H, m), 2.10-1.90 (4H, m)

¹³C-NMR (125 MHz, D₂O, δ): 178.5 (s), 176.7 (s), 174.9 (s), 174.7 (s),173.4 (s), 172.0 (s), 170.8 (s), 149.4 (s), 141.5 (s), 132.5 (s), 130.7(d), 126.5 (d), 120.2 (d), 75.7 (d), 75.0 (d), 73.5 (d), 73.0 (d), 71.9(d), 70.5 (d), 64.5 (t), 63.9 (d), 59.4 (d), 58.3 (t), 57.4 (d), 56.9(d), 54.3 (d), 48.7 (t), 41.5 (t), 41.5 (t), 39.9 (t), 35.5 (t), 31.5(t), 29.1 (t)

From the analysis of the above physical and chemical properties, and theresult of the further investigation of identification of chemicalstructure, the chemical structure of the Deacyl WF 738F has beenidentified and assigned as follows.

What is claimed is:
 1. A polypeptide compound of the following general formula (I):

wherein R¹ is hydrogen or acyl group, R² is hydrogen or hydroxy, R³ is hydrogen or methyl, and R⁴ is hydrogen or hydroxy, with proviso that when R⁴ is hydroxy, then R² is hydroxy, or a salt thereof.
 2. A compound of claim 1, wherein R¹ is hydrogen, aroyl having higher alkoxy, aroyl substituted with heterocyclic group which has aryl having lower alkoxy, aroyl substituted with heterocyclic group which has aryl having higher alkoxy, ar(lower)alkenoyl substituted with aryl having lower alkoxy, ar(lower)alkenoyl substituted with aryl having higher alkoxy, aroyl substituted with aryl which has aryl having lower alkoxy, aroyl substituted with aryl which has aryl having higher alkoxy, aroyl substituted with heterocyclic group which has aryl substituted with aryl having lower alkoxy, or aroyl substituted with heterocyclic group which has aryl substituted with aryl having higher alkoxy.
 3. A compound of claim 2, wherein R¹ is hydrogen.
 4. A process for preparing a polypeptide compound of claim 1 or a salt thereof, which comprises i) fermenting a strain belonging to the genus Coleophoma which is capable of producing a compound of the formula (Ia) or a salt thereof:

wherein R_(a) ¹ is palmitoyl and R², R³ and R⁴ are each as defined in claim 1, in a nutrient medium and recovering the compound (Ia) or a salt thereof, to give the compound (Ia) or a salt thereof; ii) subjecting a compound of (Ia) or a salt thereof, to elimination reaction of N-acyl group, to give a compound of the formula (Ib):

wherein R², R³ and R⁴ are each as defined in claim 1, or a salt thereof; or iii) subjecting a compound of (Ib) or a salt thereof thus obtained to acylation reaction, to give a compound of the formula (Ic):

wherein R_(c) ¹ is acyl group exclusive of palmitoyl, R², R³ and R⁴ are each as defined in claim 1, or a salt thereof.
 5. A pharmaceutical composition or medicament comprising the compound of claim 1 or a pharmaceutically acceptable salt thereof in admixture with a pharmaceutically acceptable carrier, diluent or excipient.
 6. An antimicrobial agent comprising the compound of claim 1, or a pharmaceutically acceptable salt thereof.
 7. An antifungal agent comprising the compound of claim 1, or a pharmaceutically acceptable salt thereof.
 8. A method for the prophylactic or the therapeutic treatment of an infectious disease caused by a pathogenic microorganism comprising administering the compound of claim 1, or a pharmaceutically acceptable salt thereof to a human being or an animal.
 9. An isolated microorganism Coleophoma crateriformis No. 738 (FERM BP-5796).
 10. A polypeptide compound or a pharmaceutically acceptable salt thereof which is obtained by culturing Coleophoma crateriformis No. 738 in a medium capable of supporting this strain.
 11. The method of claim 8, wherein said disease is a fungal disease.
 12. The method of claim 8, wherein said disease is caused by a microorganism selected from the group consisting of Aspergillus, Cyptococcus, Candida, Mucor, Actinomyces, Histoplasma, Dermatophyte, Malassezia, Fusarium and Pneumocystis.
 13. The method of claim 8, wherein said disease is caused by Pneumocystis carinii.
 14. The method of claim 8, wherein said compound is administered to a human.
 15. The composition or medicament of claim 5, selected from the group consisting of a granule, tablet, dragee, pellet, troche, capsule, suppository, cream, ointment, aerosol, powder, aerosol spray, solution, emulsion, suspension, ingestion and eye drop. 